Methods for identifying risk of breast cancer and treatments thereof

ABSTRACT

Provided herein are methods for identifying risk of breast cancer in a subject and/or a subject at risk of breast cancer, reagents and kits for carrying out the methods, methods for identifying candidate therapeutics for treating breast cancer, and therapeutic methods for treating breast cancer in a subject. These embodiments are based upon an analysis of polymorphic variations in nucleotide sequences within the human genome.

RELATED PATENT APPLICATIONS

This patent application claims priority to international application no. PCT/US2004/016942 filed May 27, 2004, having attorney docket number SEQ-4066-PC2. This patent application names Richard B. Roth et al. as inventors and is hereby incorporated herein by reference in its entirety, including all drawings and cited publications and documents.

FIELD OF THE INVENTION

The invention relates to genetic methods for identifying risk of breast cancer and treatments that specifically target the disease.

BACKGROUND

Breast cancer is the third most common cancer, and the most common cancer in women, as well as a cause of disability, psychological trauma, and economic loss. Breast cancer is the second most common cause of cancer death in women in the United States, in particular for women between the ages of 15 and 54, and the leading cause of cancer-related death (Forbes, Seminars in Oncology, vol. 24(1), Suppl 1, 1997: pp. S1-20-S1-35). Indirect effects of the disease also contribute to the mortality from breast cancer including consequences of advanced disease, such as metastases to the bone or brain. Complications arising from bone marrow suppression, radiation fibrosis and neutropenic sepsis, collateral effects from therapeutic interventions, such as surgery, radiation, chemotherapy, or bone marrow transplantation—also contribute to the morbidity and mortality from this disease.

While the pathogenesis of breast cancer is unclear, transformation of normal breast epithelium to a malignant phenotype may be the result of genetic factors, especially in women under thirty (Miki, et al., Science, 266: 66-71 (1994)). However, it is likely that other, non-genetic factors also have a significant effect on the etiology of the disease. Regardless of its origin, breast cancer morbidity increases significantly if it is not detected early in its progression. Thus, considerable efforts have focused on the elucidation of early cellular events surrounding transformation in breast tissue. Such efforts have led to the identification of several potential breast cancer markers. For example, alleles of the BRCA1 and BRCA2 genes have been linked to hereditary and early-onset breast cancer (Wooster, et al., Science, 265: 2088-2090 (1994)). However, BRCA1 is limited as a cancer marker because BRCA1 mutations fail to account for the majority of breast cancers (Ford, et al., British J. Cancer, 72: 805-812 (1995)). Similarly, the BRCA2 gene, which has been linked to forms of hereditary breast cancer, accounts for only a small portion of total breast cancer cases.

SUMMARY

It has been discovered that certain polymorphic variations in human genomic DNA are associated with the occurrence of breast cancer. Thus, featured herein are methods for identifying a subject at risk of breast cancer and/or a risk of breast cancer in a subject, which comprises detecting the presence or absence of one or more of the polymorphic variations described herein in a human nucleic acid sample. Also featured herein are nucleic acids that include one or more polymorphic variations associated with the occurrence of breast cancer, as well as polypeptides encoded by these nucleic acids. Further, provided is a method for identifying a subject at risk of breast cancer and then prescribing to the subject a breast cancer detection procedure, prevention procedure and/or a treatment procedure. In addition, provided are methods for identifying candidate therapeutic molecules for treating breast cancer and related disorders, as well as methods for treating breast cancer in a subject by diagnosing breast cancer in the subject and treating the subject with a suitable treatment, such as administering a therapeutic molecule.

Also, featured is a method for inhibiting metastasis of breast cancer cells into other tissues, which comprises inhibiting a KIAA0861 nucleic acid or substantially identical nucleic acid thereof (e.g., reducing the amount of polypeptide expressed from mRNA encoded by the nucleotide sequence), or inhibiting a KIAA0861 polypeptide or substantially identical polypeptide thereof (e.g., inhibiting the guanine nucleotide exchange function of the KIAA0861 polypeptide). The inhibition is effected by contacting a system with a molecule having the inhibitory activity, where the system sometimes is a group of cells in vitro, a tissue sample in vitro, or an animal such as a human, often a female. In an embodiment, the KIAA0861 nucleic acid or substantially identical nucleic acid thereof is inhibited by contacting cells overexpressing the KIAA0861 nucleotide sequence with an RNA molecule, and in certain embodiments, the RNA molecule is double stranded with one strand complementary to a subsequence of the KIAA0861 nucleotide sequence.

Also provided are compositions comprising a breast cancer cell and/or a KIAA0861 nucleic acid with a RNAi, siRNA, antisense DNA or RNA, or ribozyme nucleic acid designed from a KIAA0861 nucleotide sequence. In an embodiment, the nucleic acid is designed from a KIAA0861 nucleotide sequence that includes one or more breast cancer associated polymorphic variations, and in some instances, specifically interacts with such a nucleotide sequence. Further, provided are arrays of nucleic acids bound to a solid surface, in which one or more nucleic acid molecules of the array have a KIAA0861 nucleotide sequence, or a fragment or substantially identical nucleic acid thereof, or a complementary nucleic acid of the foregoing. Featured also are compositions comprising a breast cancer cell and/or a KIAA0861 polypeptide, with an antibody that specifically binds to the polypeptide. In an embodiment, the antibody specifically binds to an epitope in the polypeptide that includes a non-synonymous amino acid modification associated with breast cancer (e.g., results in an amino acid substitution in the encoded polypeptide associated with breast cancer). In certain embodiments, the antibody specifically binds to an epitope that comprises a leucine at amino acid position 276 in SEQ ID NO: 4, a leucine at amino acid position 295 in SEQ ID NO: 4, a phenylalanine at amino acid position 506 in SEQ ID NO: 4, or an alanine at amino acid position 819 in SEQ ID NO: 4. Alternatively, the antibody specifically binds to an epitope that comprises a leucine at amino acid position 359 in SEQ ID NO: 5, a leucine at amino acid position 378 in SEQ ID NO: 5, a phenylalanine at amino acid position 589 in SEQ ID NO: 4, or an alanine at amino acid position 902 in SEQ ID NO: 5.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows proximal SNPs in and around a KIAA0861 region. The position of each SNP on the chromosome is shown on the x-axis and the y-axis provides the negative logarithm of the p-value comparing the estimated allele to that of the control group. Also shown in the figure are exons and introns of the region in the approximate chromosomal positions. The figure indicates that polymorphic variants associated with breast cancer are in a region spanning chromosome positions 184215647 to 184249849 on chromosome 3 (based on NCBI's Build 34).

DETAILED DESCRIPTION

It has been discovered that a polymorphic variation in a gene encoding a novel member of the DBL family of Rho guanine nucleotide exchange factors (RhoGEFs), known as KIAA0861, is associated with the occurrence of breast cancer. DBL RhoGEF proteins are characterized by two distinct domains, the Dbl homology (DH) domain and the pleckstrin homology (PH) domain, which are believed to be responsible for catalyzing the GDP-GTP exchange reaction of Rho proteins. RhoGEFs bind to the GDP-bound form and destabilize GDP-RhoGTPases while stabilizing a nucleotide-free reaction intermediate. Because of the high intracellular ratio of GTP:GDP, the released GTP is replaced with GTP, leading to activation. Rho family GTP-binding proteins (Rho GTPases) belong to the Ras-related G protein superfamily and function in controlling numerous cellular activities, including cell growth, adhesion, and movement. The Rho GTPase family includes members RhoA, RacI and Cdc42, which stimulate the cyclin D1 promoter and cause upregulation of cyclin D1 protein. RacI and Cdc42 promote inactivation of Rb and stimulation of E2F-mediated transcription. Signaling pathways for RhoGEFs and RhoGTPases are set forth in Schmidt & Hall, Genes and Development 16: 1587-1609 (2002) and Pruitt & Der, Cancer Letters 171: 1-10 (2001).

KIAA0861 shares strong homology with members of the Dbl family of Rho guanine nucleotide exchange factors (RhoGEFs), a family of over 60 proteins that function by catalyzing the exchange of Rho-bound GDP for GTP. Rho family GTP-binding proteins (Rho GTPases) belong to the Ras-related G protein superfamily and function in controlling numerous cellular activities, including cell growth, adhesion and movement. Over 18 Ras-related G protein superfamily members have been described to date, including several Rho-family GTP-binding proteins (Rho GTPases). Rho GTPases are membrane bound molecular switches that are active when bound to GTP and inactive when bound to GDP. Deregulation of both Rho GTPase activity and RhoGEF activity have been shown to be oncogenic. Several studies have shown that deregulation of Rho GTPase activity leads to loss of contact inhibition, growth factor dependence and anchorage dependence in a variety of cell types (Whitehead, I P, et al (1997) Biochem. Biophys. Acta, 1332: F1-F23).

Deregulation of both Rho GTPase activity and RhoGEF activity have been shown to be oncogenic. For example, DBS, a Rho-specific guanine nucleotide exchange factor (RhoGEF), exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts (Cheng, L, et al. (2002) MCB 22 (19):6895-6905). Several studies have shown that deregulation of Rho GTPase activity leads to loss of contact inhibition, growth factor dependence and anchorage dependence in a variety of cell types. Further, recent evidence has shown that deregulation of RhoGEF activity results in tumorigenic growth and promotes invasive potential (Whitehead, I P, et al. (1997) Biochem. Biophys. Acta, 1332: F1-F23). Interestingly, cellular transformation via deregulated RhoGEF function is much stronger than through deregulation of Rho GTPases (Lin, R, Cerione, R A, and Manor, D (1999) JBC, 274: 23633-23641).

Breast Cancer and Sample Selection

Breast cancer is typically described as the uncontrolled growth of malignant breast tissue. Breast cancers arise most commonly in the lining of the milk ducts of the breast (ductal carcinoma), or in the lobules where breast milk is produced (lobular carcinoma). Other forms of breast cancer include Inflammatory Breast Cancer and Recurrent Breast Cancer. Inflammatory breast cancer is a rare, but very serious, aggressive type of breast cancer. The breast may look red and feel warm with ridges, welts, or hives on the breast; or the skin may look wrinkled. It is sometimes misdiagnosed as a simple infection. Recurrent disease means that the cancer has come back after it has been treated. It may come back in the breast, in the soft tissues of the chest (the chest wall), or in another part of the body.

As used herein, the term “breast cancer” refers to a condition characterized by anomalous rapid proliferation of abnormal cells in one or both breasts of a subject. The abnormal cells often are referred to as “neoplastic cells,” which are transformed cells that can form a solid tumor. The term “tumor” refers to an abnormal mass or population of cells (i.e. two or more cells) that result from excessive or abnormal cell division, whether malignant or benign, and pre-cancerous and cancerous cells. Malignant tumors are distinguished from benign growths or tumors in that, in addition to uncontrolled cellular proliferation, they can invade surrounding tissues and can metastasize. In breast cancer, neoplastic cells may be identified in one or both breasts only and not in another tissue or organ, in one or both breasts and one or more adjacent tissues or organs (e.g. lymph node), or in a breast and one or more non-adjacent tissues or organs to which the breast cancer cells have metastasized.

The term “invasion” as used herein refers to the spread of cancerous cells to adjacent surrounding tissues. The term “invasion” often is used synonymously with the term “metastasis,” which as used herein refers to a process in which cancer cells travel from one organ or tissue to another non-adjacent organ or tissue. Cancer cells in the breast(s) can spread to tissues and organs of a subject, and conversely, cancer cells from other organs or tissue can invade or metastasize to a breast. Cancerous cells from the breast(s) may invade or metastasize to any other organ or tissue of the body. Breast cancer cells often invade lymph node cells and/or metastasize to the liver, brain and/or bone and spread cancer in these tissues and organs. Breast cancers can spread to other organs and tissues and cause lung cancer, prostate cancer, colon cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, bladder cancer, hepatoma, colorectal cancer, uterine cervical cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, vulval cancer, thyroid cancer, hepatic carcinoma, skin cancer, melanoma, ovarian cancer, neuroblastoma, myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma and peripheral neuroepithelioma, and other carcinomas, lymphomas, blastomas, sarcomas, and leukemias.

Breast cancers arise most commonly in the lining of the milk ducts of the breast (ductal carcinoma), or in the lobules where breast milk is produced (lobular carcinoma). Other forms of breast cancer include Inflammatory Breast Cancer and Recurrent Breast Cancer. Inflammatory Breast Cancer is a rare, but very serious, aggressive type of breast cancer. The breast may look red and feel warm with ridges, welts, or hives on the breast; or the skin may look wrinkled. It is sometimes misdiagnosed as a simple infection. Recurrent disease means that the cancer has come back after it has been treated. It may come back in the breast, in the soft tissues of the chest (the chest wall), or in another part of the body. As used herein, the term “breast cancer” may include both Inflammatory Breast Cancer and Recurrent Breast Cancer.

In an effort to detect breast cancer as early as possible, regular physical exams and screening mammograms often are prescribed and conducted. A diagnostic mammogram often is performed to evaluate a breast complaint or abnormality detected by physical exam or routine screening mammography. If an abnormality seen with diagnostic mammography is suspicious, additional breast imaging (with exams such as ultrasound) or a biopsy may be ordered. A biopsy followed by pathological (microscopic) analysis is a definitive way to determine whether a subject has breast cancer. Excised breast cancer samples often are subjected to the following analyses: diagnosis of the breast tumor and confirmation of its malignancy; maximum tumor thickness; assessment of completeness of excision of invasive and in situ components and microscopic measurements of the shortest extent of clearance; level of invasion; presence and extent of regression; presence and extent of ulceration; histological type and special variants; pre-existing lesion; mitotic rate; vascular invasion; neurotropism; cell type; tumor lymphocyte infiltration; and growth phase.

The stage of a breast cancer can be classified as a range of stages from Stage 0 to Stage IV based on its size and the extent to which it has spread. The following table summarizes the stages:

TABLE A Metastasis Stage Tumor Size Lymph Node Involvement (Spread) I Less than 2 cm No No II Between 2-5 cm No or in same side of breast No III More than 5 cm Yes, on same side of breast No IV Not applicable Not applicable Yes

Stage 0 cancer is a contained cancer that has not spread beyond the breast ductal system. Fifteen to twenty percent of breast cancers detected by clinical examinations or testing are in Stage 0 (the earliest form of breast cancer). Two types of Stage 0 cancer are lobular carcinoma in situ (LCIS) and ductal carcinoma in situ (DCIS). LCIS indicates high risk for breast cancer. Many physicians do not classify LCIS as a malignancy and often encounter LCIS by chance on breast biopsy while investigating another area of concern. While the microscopic features of LCIS are abnormal and are similar to malignancy, LCIS does not behave as a cancer (and therefore is not treated as a cancer). LCIS is merely a marker for a significantly increased risk of cancer anywhere in the breast. However, bilateral simple mastectomy may be occasionally performed if LCIS patients have a strong family history of breast cancer. In DCIS the cancer cells are confined to milk ducts in the breast and have not spread into the fatty breast tissue or to any other part of the body (such as the lymph nodes). DCIS may be detected on mammogram as tiny specks of calcium (known as microcalcifications) 80% of the time. Less commonly DCIS can present itself as a mass with calcifications (15% of the time); and even less likely as a mass without calcifications (<5% of the time). A breast biopsy is used to confirm DCIS. A standard DCIS treatment is breast-conserving therapy (BCT), which is lumpectomy followed by radiation treatment or mastectomy. To date, DCIS patients have chosen equally among lumpectomy and mastectomy as their treatment option, though specific cases may sometimes favor lumpectomy over mastectomy or vice versa.

In Stage I, the primary (original) cancer is 2 cm or less in diameter and has not spread to the lymph nodes. In Stage IIA, the primary tumor is between 2 and 5 cm in diameter and has not spread to the lymph nodes. In Stage IIB, the primary tumor is between 2 and 5 cm in diameter and has spread to the axillary (underarm) lymph nodes; or the primary tumor is over 5 cm and has not spread to the lymph nodes. In Stage IIIA, the primary breast cancer of any kind that has spread to the axillary (underarm) lymph nodes and to axillary tissues. In Stage IIIB, the primary breast cancer is any size, has attached itself to the chest wall, and has spread to the pectoral (chest) lymph nodes. In Stage IV, the primary cancer has spread out of the breast to other parts of the body (such as bone, lung, liver, brain). The treatment of Stage IV breast cancer focuses on extending survival time and relieving symptoms.

Based in part upon selection criteria set forth above, individuals having breast cancer can be selected for genetic studies. Also, individuals having no history of cancer or breast cancer often are selected for genetic studies. Other selection criteria can include: a tissue or fluid sample is derived from an individual characterized as Caucasian; the sample was derived from an individual of German paternal and maternal descent; the database included relevant phenotype information for the individual; case samples were derived from individuals diagnosed with breast cancer; control samples were derived from individuals free of cancer and no family history of breast cancer; and sufficient genomic DNA was extracted from each blood sample for all allelotyping and genotyping reactions performed during the study. Phenotype information included pre- or post-menopausal, familial predisposition, country or origin of mother and father, diagnosis with breast cancer (date of primary diagnosis, age of individual as of primary diagnosis, grade or stage of development, occurrence of metastases, e.g., lymph node metastases, organ metastases), condition of body tissue (skin tissue, breast tissue, ovary tissue, peritoneum tissue and myometrium), method of treatment (surgery, chemotherapy, hormone therapy, radiation therapy).

Provided herein is a set of blood samples and a set of corresponding nucleic acid samples isolated from the blood samples, where the blood samples are donated from individuals diagnosed with breast cancer. The sample set often includes blood samples or nucleic acid samples from 100 or more, 150 or more, or 200 or more individuals having breast cancer, and sometimes from 250 or more, 300 or more, 400 or more, or 500 or more individuals. The individuals can have parents from any place of origin, and in an embodiment, the set of samples are extracted from individuals of German paternal and German maternal ancestry. The samples in each set may be selected based upon five or more criteria and/or phenotypes set forth above.

Polymorphic Variants Associated with Breast Cancer

A genetic analysis provided herein linked breast cancer with polymorphic variants in and around a nucleotide sequence located on chromosome three that encodes a Rho family guanine-nucleotide exchange factor polypeptide designated KIAA0861. As used herein, the term “polymorphic site” refers to a region in a nucleic acid at which two or more alternative nucleotide sequences are observed in a significant number of nucleic acid samples from a population of individuals. A polymorphic site may be a nucleotide sequence of two or more nucleotides, an inserted nucleotide or nucleotide sequence, a deleted nucleotide or nucleotide sequence, or a microsatellite, for example. A polymorphic site that is two or more nucleotides in length may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, 20 or more, 30 or more, 50 or more, 75 or more, 100 or more, 500 or more, or about 1000 nucleotides in length, where all or some of the nucleotide sequences differ within the region. A polymorphic site is often one nucleotide in length, which is referred to herein as a “single nucleotide polymorphism” or a “SNP.”

Where there are two, three, or four alternative nucleotide sequences at a polymorphic site, each nucleotide sequence is referred to as a “polymorphic variant” or “nucleic acid variant.” Where two polymorphic variants exist, for example, the polymorphic variant represented in a minority of samples from a population is sometimes referred to as a “minor allele” and the polymorphic variant that is more prevalently represented is sometimes referred to as a “major allele.” Many organisms possess a copy of each chromosome (e.g., humans), and those individuals who possess two major alleles or two minor alleles are often referred to as being “homozygous” with respect to the polymorphism, and those individuals who possess one major allele and one minor allele are normally referred to as being “heterozygous” with respect to the polymorphism. Individuals who are homozygous with respect to one allele are sometimes predisposed to a different phenotype as compared to individuals who are heterozygous or homozygous with respect to another allele.

Furthermore, a genotype or polymorphic variant may be expressed in terms of a “haplotype,” which as used herein refers to two or more polymorphic variants occurring within genomic DNA in a group of individuals within a population. For example, two SNPs may exist within a gene where each SNP position includes a cytosine variation and an adenine variation. Certain individuals in a population may carry one allele (heterozygous) or two alleles (homozygous) having the gene with a cytosine at each SNP position. As the two cytosines corresponding to each SNP in the gene travel together on one or both alleles in these individuals, the individuals can be characterized as having a cytosine/cytosine haplotype with respect to the two SNPs in the gene.

As used herein, the term “phenotype” refers to a trait which can be compared between individuals, such as presence or absence of a condition, a visually observable difference in appearance between individuals, metabolic variations, physiological variations, variations in the function of biological molecules, and the like. An example of a phenotype is occurrence of breast cancer.

Researchers sometimes report a polymorphic variant in a database without determining whether the variant is represented in a significant fraction of a population. Because a subset of these reported polymorphic variants are not represented in a statistically significant portion of the population, some of them are sequencing errors and/or not biologically relevant. Thus, it is often not known whether a reported polymorphic variant is statistically significant or biologically relevant until the presence of the variant is detected in a population of individuals and the frequency of the variant is determined. Methods for detecting a polymorphic variant in a population are described herein, specifically in Example 2. A polymorphic variant is statistically significant and often biologically relevant if it is represented in 5% or more of a population, sometimes 10% or more, 15% or more, or 20% or more of a population, and often 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more of a population.

A polymorphic variant may be detected on either or both strands of a double-stranded nucleic acid. For example, a thymine at a particular position in SEQ ID NO: 1 can be reported as an adenine from the complementary strand. Also, a polymorphic variant may be located within an intron or exon of a gene or within a portion of a regulatory region such as a promoter, a 5′ untranslated region (UTR), a 3′ UTR, and in DNA (e.g., genomic DNA (gDNA) and complementary DNA (cDNA)), RNA (e.g., mRNA, tRNA, and rRNA), or a polypeptide. Polymorphic variations may or may not result in detectable differences in gene expression, polypeptide structure, or polypeptide function.

In the genetic analysis that associated breast cancer with the polymorphic variants described hereafter, samples from individuals having breast cancer and individuals not having cancer were allelotyped and genotyped. The term “genotyped” as used herein refers to a process for determining a genotype of one or more individuals, where a “genotype” is a representation of one or more polymorphic variants in a population. Genotypes may be expressed in terms of a “haplotype,” which as used herein refers to two or more polymorphic variants occurring within genomic DNA in a group of individuals within a population. For example, two SNPs may exist within a gene where each SNP position includes a cytosine variation and an adenine variation. Certain individuals in a population may carry one allele (heterozygous) or two alleles (homozygous) having the gene with a cytosine at each SNP position. As the two cytosines corresponding to each SNP in the gene travel together on one or both alleles in these individuals, the individuals can be characterized as having a cytosine/cytosine haplotype with respect to the two SNPs in the gene.

It was determined that polymorphic variations associated with an increased risk of breast cancer existed in KIAA0861 nucleotide sequences. Polymorphic variants in and around the KIAA0861 locus were tested for an association with breast cancer. These included polymorphic variants at positions selected from the group consisting of rs3811728, rs3811729, rs602646, rs488277, rs1629673, rs670232, rs575326, rs575386, rs684846, rs471365, rs496251, rs831246, rs831247, rs512071, rs1502761, rs681516, rs683302, rs619424, rs620722, rs529055, rs664010, rs678454, rs2653845, rs472795, rs507079, rs534333, rs535298, rs536213, rs831245, rs639690, rs684174, rs571761, rs1983421, rs4630966, rs2314415, rs6788196, rs2103062, rs9827084, rs9864865, rs6804951, rs6770548, rs1403452, rs7609994, rs9838250, rs9863404, rs903950, rs6787284, rs2017340, rs2001449, rs1317288, rs7635891, rs10704581, rs11371910, rs10937118, rs7642053, rs3821522, rs2029926, rs1390831, rs7643890, rs11925606, rs9826325, rs6800429, rs6803368, rs1353566, rs2272115, rs2272116, rs3732603, rs940055, rs2314730, rs2030578, rs2049280, rs3732602, rs2293203 and rs7639705; and position 13507 of SEQ ID NO: 1. These positions correspond to positions 246, 393, 628, 7586, 9223, 9933, 10154, 10175, 10877, 10907, 11289, 11793, 11813, 14249, 14586, 14647, 15004, 16573, 16811, 18921, 19651, 20565, 25239, 25721, 27133, 27778, 27906, 28000, 30005, 30520, 32195, 32439, 33858, 41716, 42450, 43554, 44211, 44775, 44962, 45317, 45712, 45941, 46520, 47175, 48045, 48636, 48689, 48704, 48849, 48850, 49931, 51510, 51526, 51758, 51975, 53475, 55524, 56754, 57473, 57497, 57613, 58023, 58821, 59644, 66217, 66344, 67326, 69777, 83594, 84579, 85623 and 13507 in SEQ ID NO: 1, respectively. Polymorphic variants in a region spanning positions 14647 to 48849 in SEQ ID NO: 1 were in particular associated with an increased risk of breast cancer, including polymorphic variants at positions 41716, 44775, 44962, 45317, 45712, 45941, and 48849 in SEQ ID NO: 1 (i.e., positions designated by rs4630966, rs9827084, rs9864865, rs6804951, rs6770548, rs1403452 and rs2001449, respectively). At these positions in SEQ ID NO: 1, a cytosine at position 41716, a guanine at position 44775, a guanine at position 44962, a cytosine at position 45317, a guanine at position 45712, a thymine at position 45941, and a cytosine at position 48849 were in particular associated with an increased risk of breast cancer. Also, an alanine at amino acid position 819 in SEQ ID NO: 4 (or position 902 in SEQ ID NO: 5) was in particular associated with an increased risk of breast cancer.

Based in part upon analyses summarized in FIG. 1, a region with significant association has been identified in a KIAA0861 region associated with increased risk of breast cancer. Any polymorphic variants associated with an increased risk of breast cancer in a region of significant association can be utilized for embodiments described herein. The following reports such a region, where “begin” and “end” designate the boundaries of the region according to chromosome positions within NCBI's Genome build 34. The chromosome on which the KIAA0861 region resides and an incident polymorphism in the locus also are noted.

Incident chr begin End size 2001449 3 184215647 184249849 34202 The polymorphic variants described above and in the Examples section are applicable to embodiments described hereafter.

Additional Polymorphic Variants Associated with Breast Cancer

Also provided is a method for identifying polymorphic variants proximal to an incident, founder polymorphic variant associated with breast cancer. Thus, featured herein are methods for identifying a polymorphic variation associated with breast cancer that is proximal to an incident polymorphic variation associated with breast cancer, which comprises identifying a polymorphic variant proximal to the incident polymorphic variant associated with breast cancer, where the incident polymorphic variant is in a nucleotide sequence set forth in SEQ ID NO: 1. The nucleotide sequence often comprises a polynucleotide sequence selected from the group consisting of (a) a nucleotide sequence set forth in SEQ ID NO: 1; (b) a nucleotide sequence which encodes a polypeptide having an amino acid sequence encoded by a nucleotide sequence in SEQ ID NO: 1; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence in SEQ ID NO: 1 or a nucleotide sequence about 90% or more identical to the nucleotide sequence set forth in SEQ ID NO: 1; and (d) a fragment of a nucleotide sequence of (a), (b), or (c), often a fragment that includes a polymorphic site associated with breast cancer. The presence or absence of an association of the proximal polymorphic variant with breast cancer then is determined using a known association method, such as a method described in the Examples hereafter. In an embodiment, the incident polymorphic variant is set forth in SEQ ID NO: 1. In another embodiment, the proximal polymorphic variant identified sometimes is a publicly disclosed polymorphic variant, which for example, sometimes is published in a publicly available database. In other embodiments, the polymorphic variant identified is not publicly disclosed and is discovered using a known method, including, but not limited to, sequencing a region surrounding the incident polymorphic variant in a group of nucleic samples. Thus, multiple polymorphic variants proximal to an incident polymorphic variant are associated with breast cancer using this method.

The proximal polymorphic variant often is identified in a region surrounding the incident polymorphic variant. In certain embodiments, this surrounding region is about 50 kb flanking the first polymorphic variant (e.g. about 50 kb 5′ of the first polymorphic variant and about 50 kb 3′ of the first polymorphic variant), and the region sometimes is composed of shorter flanking sequences, such as flanking sequences of about 40 kb, about 30 kb, about 25 kb, about 20 kb, about 15 kb, about 10 kb, about 7 kb, about 5 kb, or about 2 kb 5′ and 3′ of the incident polymorphic variant. In other embodiments, the region is composed of longer flanking sequences, such as flanking sequences of about 55 kb, about 60 kb, about 65 kb, about 70 kb, about 75 kb, about 80 kb, about 85 kb, about 90 kb, about 95 kb, or about 100 kb 5′ and 3′ of the incident polymorphic variant.

In certain embodiments, polymorphic variants associated with breast cancer are identified iteratively. For example, a first proximal polymorphic variant is associated with breast cancer using the methods described above and then another polymorphic variant proximal to the first proximal polymorphic variant is identified (e.g., publicly disclosed or discovered) and the presence or absence of an association of one or more other polymorphic variants proximal to the first proximal polymorphic variant with breast cancer is determined.

The methods described herein are useful for identifying or discovering additional polymorphic variants that may be used to further characterize a gene, region or loci associated with a condition, a disease (e.g., breast cancer), or a disorder. For example, allelotyping or genotyping data from the additional polymorphic variants may be used to identify a functional mutation or a region of linkage disequilibrium.

In certain embodiments, polymorphic variants identified or discovered within a region comprising the first polymorphic variant associated with breast cancer are genotyped using the genetic methods and sample selection techniques described herein, and it can be determined whether those polymorphic variants are in linkage disequilibrium with the first polymorphic variant. The size of the region in linkage disequilibrium with the first polymorphic variant also can be assessed using these genotyping methods. Thus, provided herein are methods for determining whether a polymorphic variant is in linkage disequilibrium with a first polymorphic variant associated with breast cancer, and such information can be used in prognosis methods described herein.

Isolated KIAA0861 Nucleic Acids

Featured herein are isolated KIAA0861 nucleic acids, which include the nucleic acid having the nucleotide sequence of SEQ ID NO: 1, 2 or 3, nucleic acid variants, and substantially identical nucleic acids of the foregoing. Nucleotide sequences of the KIAA0861 nucleic acids sometimes are referred to herein as “KIAA0861 nucleotide sequences.” A “KIAA0861 nucleic acid variant” refers to one allele that may have one or more different polymorphic variations as compared to another allele in another subject or the same subject. A polymorphic variation in the KIAA0861 nucleic acid variant may be represented on one or both strands in a double-stranded nucleic acid or on one chromosomal complement (heterozygous) or both chromosomal complements (homozygous).

As used herein, the term “nucleic acid” includes DNA molecules (e.g., a complementary DNA (cDNA) and genomic DNA (gDNA)) and RNA molecules (e.g., mRNA, rRNA, and tRNA) and analogs of DNA or RNA, for example, by use of nucleotide analogs. The nucleic acid molecule can be single-stranded and it is often double-stranded. The term “isolated or purified nucleic acid” refers to nucleic acids that are separated from other nucleic acids present in the natural source of the nucleic acid. For example, with regard to genomic DNA, the term “isolated” includes nucleic acids which are separated from the chromosome with which the genomic DNA is naturally associated. An “isolated” nucleic acid is often free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences which flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. As used herein, the term “KIAA0861 gene” refers to a nucleotide sequence that encodes a KIAA0861 polypeptide.

In particular embodiments, a nucleic acid comprises a polymorphic variation corresponding to position 13507 of SEQ ID NO: 1. The nucleic acid often comprises a part of or all of a nucleotide sequence in SEQ ID NO: 1, 2 and/or 3, or a substantially identical sequence thereof and sometimes such a nucleotide sequence is a 5′ and/or 3′ sequence flanking a polymorphic variant described above that is 5, 6, 7 . . . 50, 51, 52 . . . 100, 101, 102 . . . 500, 501, 502 . . . 999 or 1000 nucleotides in length. Other embodiments are directed to methods of identifying a polymorphic variation at one or more positions in a nucleic acid (e.g., genotyping at one or more positions in the nucleic acid), where a position corresponds to position 13507 of SEQ ID NO: 1.

Also included herein are nucleic acid fragments. These fragments typically are a nucleotide sequence identical to a nucleotide sequence in SEQ ID NO: 1, 2 or 3, a nucleotide sequence substantially identical to a nucleotide sequence in SEQ ID NO: 1, 2 or 3, or a nucleotide sequence that is complementary to the foregoing. The nucleic acid fragment may be identical, substantially identical or homologous to a nucleotide sequence in an exon or an intron in SEQ ID NO: 1, and may encode a domain or part of a domain or motif of a KIAA0861 polypeptide. Domains and motifs of a KIAA0861 polypeptide include, but are not limited to, a Sec 14p-like lipid binding domain, spectrin repeats (SPEC), a RhoGEF domain (also called the DBL-homology domain (DH domain)), and a Pleckstrin-homology domain (PH domain). Sometimes, the fragment will comprises the polymorphic variation described herein as being associated with breast cancer. The nucleic acid fragment sometimes is 50, 100, or 200 or fewer base pairs in length, and is sometimes about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3800, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 110000, 120000, 130000, 140000, 150000 or 160000 base pairs in length. A nucleic acid fragment complementary to a nucleotide sequence identical or substantially identical to the nucleotide sequence of SEQ ID NO: 1, 2 or 3 and hybridizes to such a nucleotide sequence under stringent conditions often is referred to as a “probe.” Nucleic acid fragments often include one or more polymorphic sites, or sometimes have an end that is adjacent to a polymorphic site as described hereafter.

An example of a nucleic acid fragment is an oligonucleotide. As used herein, the term “oligonucleotide” refers to a nucleic acid comprising about 8 to about 50 covalently linked nucleotides, often comprising from about 8 to about 35 nucleotides, and more often from about 10 to about 25 nucleotides. The backbone and nucleotides within an oligonucleotide may be the same as those of naturally occurring nucleic acids, or analogs or derivatives of naturally occurring nucleic acids, provided that oligonucleotides having such analogs or derivatives retain the ability to hybridize specifically to a nucleic acid comprising a targeted polymorphism. Oligonucleotides described herein may be used as hybridization probes or as components of prognostic or diagnostic assays, for example, as described herein.

Oligonucleotides are typically synthesized using standard methods and equipment, such as the ABI 3900 High Throughput DNA Synthesizer and the EXPEDITE™ 8909 Nucleic Acid Synthesizer, both of which are available from Applied Biosystems (Foster City, Calif.). Analogs and derivatives are exemplified in U.S. Pat. Nos. 4,469,863; 5,536,821; 5,541,306; 5,637,683; 5,637,684; 5,700,922; 5,717,083; 5,719,262; 5,739,308; 5,773,601; 5,886,165; 5,929,226; 5,977,296; 6,140,482; WO 00/56746; WO 01/14398, and related publications. Methods for synthesizing oligonucleotides comprising such analogs or derivatives are disclosed, for example, in the patent publications cited above and in U.S. Pat. Nos. 5,614,622; 5,739,314; 5,955,599; 5,962,674; 6,117,992; in WO 00/75372; and in related publications.

Oligonucleotides also may be linked to a second moiety. The second moiety may be an additional nucleotide sequence such as a tail sequence (e.g., a polyadenosine tail), an adapter sequence (e.g., phage M13 universal tail sequence), and others. Alternatively, the second moiety may be a non-nucleotide moiety such as a moiety which facilitates linkage to a solid support or a label to facilitate detection of the oligonucleotide. Such labels include, without limitation, a radioactive label, a fluorescent label, a chemiluminescent label, a paramagnetic label, and the like. The second moiety may be attached to any position of the oligonucleotide, provided the oligonucleotide can hybridize to the nucleic acid comprising the polymorphism.

Uses for Nucleic Acid Sequences

Nucleic acid coding sequences depicted in SEQ ID NO: 2 or 3 may be used for diagnostic purposes for detection and control of polypeptide expression. Also, included herein are oligonucleotide sequences such as antisense RNA, small-interfering RNA (siRNA) and DNA molecules and ribozymes that function to inhibit translation of a polypeptide. Antisense techniques and RNA interference techniques are known in the art and are described herein.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage. Ribozymes may be engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences corresponding to or complementary to the nucleotide sequences set forth in SEQ ID NO: 1-3. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between fifteen (15) and twenty (20) ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.

Antisense RNA and DNA molecules, siRNA and ribozymes may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

DNA encoding a polypeptide also may have a number of uses for the diagnosis of diseases, including breast cancer, resulting from aberrant expression of a target gene described herein. For example, the nucleic acid sequence may be used in hybridization assays of biopsies or autopsies to diagnose abnormalities of expression or function (e.g., Southern or Northern blot analysis, in situ hybridization assays).

In addition, the expression of a polypeptide during embryonic development may also be determined using nucleic acid encoding the polypeptide. As addressed, infra, production of functionally impaired polypeptide can be the cause of various disease states, such as breast cancer. In situ hybridizations using polynucleotide probes may be employed to predict problems related to breast cancer. Further, as indicated, infra, administration of human active polypeptide, recombinantly produced as described herein, may be used to treat disease states related to functionally impaired polypeptide (e.g., a KIAA0861 polypeptide that activates a Rho GTPase in a situation where it is not normally activated). Alternatively, gene therapy approaches may be employed to remedy deficiencies of functional polypeptide or to replace or compete with dysfunctional polypeptide.

Expression Vectors, Host Cells, and Genetically Engineered Cells

Provided herein are nucleic acid vectors, often expression vectors, which contain a KIAA0861 nucleic acid. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid, or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors may include replication defective retroviruses, adenoviruses and adeno-associated viruses for example.

A vector can include a KIAA0861 nucleic acid in a form suitable for expression of the nucleic acid in a host cell. The recombinant expression vector typically includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term “regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, and the like. Expression vectors can be introduced into host cells to produce KIAA0861 polypeptides, including fusion polypeptides, encoded by KIAA0861 nucleic acids.

Recombinant expression vectors can be designed for expression of KIAA0861 polypeptides in prokaryotic or eukaryotic cells. For example, KIAA0861 polypeptides can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of polypeptides in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant polypeptide; 2) to increase the solubility of the recombinant polypeptide; and 3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith & Johnson, Gene 67: 31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.

Purified fusion polypeptides can be used in screening assays and to generate antibodies specific for KIAA0861 polypeptides. In a therapeutic embodiment, fusion polypeptide expressed in a retroviral expression vector is used to infect bone marrow cells that are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks).

Expressing the polypeptide in host bacteria with an impaired capacity to proteolytically cleave the recombinant polypeptide is often used to maximize recombinant polypeptide expression (Gottesman, S., Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. 185: 119-128 (1990)). Another strategy is to alter the nucleotide sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., Nucleic Acids Res. 20: 2111-2118 (1992)). Such alteration of nucleotide sequences can be carried out by standard DNA synthesis techniques.

When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. Recombinant mammalian expression vectors are often capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue-specific promoters include an albumin promoter (liver-specific; Pinkert et al., Genes Dev. 1: 268-277 (1987)), lymphoid-specific promoters (Calame & Eaton, Adv. Immunol. 43: 235-275 (1988)), promoters of T cell receptors (Winoto & Baltimore, EMBO J. 8: 729-733 (1989)) promoters of immunoglobulins (Banerji et al., Cell 33: 729-740 (1983); Queen & Baltimore, Cell 33: 741-748 (1983)), neuron-specific promoters (e.g., the neurofilament promoter; Byrne & Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477 (1989)), pancreas-specific promoters (Edlund et al., Science 230: 912-916 (1985)), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are sometimes utilized, for example, the murine hox promoters (Kessel & Gruss, Science 249: 374-379 (1990)) and the α-fetopolypeptide promoter (Campes & Tilghman, Genes Dev. 3: 537-546 (1989)).

A KIAA0861 nucleic acid may also be cloned into an expression vector in an antisense orientation. Regulatory sequences (e.g., viral promoters and/or enhancers) operatively linked to a KIAA0861 nucleic acid cloned in the antisense orientation can be chosen for directing constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types. Antisense expression vectors can be in the form of a recombinant plasmid, phagemid or attenuated virus. For a discussion of the regulation of gene expression using antisense genes see Weintraub et al., Antisense RNA as a molecular tool for genetic analysis, Reviews—Trends in Genetics, Vol. 1(1) (1986).

Also provided herein are host cells that include a KIAA0861 nucleic acid within a recombinant expression vector or KIAA0861 nucleic acid sequence fragments which allow it to homologously recombine into a specific site of the host cell genome. The terms “host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but rather also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic or eukaryotic cell. For example, a KIAA0861 polypeptide can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

Vectors can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, transduction/infection, DEAE-dextran-mediated transfection, lipofection, or electroporation.

A host cell provided herein can be used to produce (i.e., express) a KIAA0861 polypeptide. Accordingly, further provided are methods for producing a KIAA0861 polypeptide using the host cells described herein. In one embodiment, the method includes culturing host cells into which a recombinant expression vector encoding a KIAA0861 polypeptide has been introduced in a suitable medium such that a KIAA0861 polypeptide is produced. In another embodiment, the method further includes isolating a KIAA0861 polypeptide from the medium or the host cell.

Also provided are cells or purified preparations of cells which include a KIAA0861 transgene, or which otherwise misexpress KIAA0861 polypeptide. Cell preparations can consist of human or non-human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In certain embodiments, the cell or cells include a KIAA0861 transgene (e.g., a heterologous form of a KIAA0861 such as a human gene expressed in non-human cells). The KIAA0861 transgene can be misexpressed, e.g., overexpressed or underexpressed. In other embodiments, the cell or cells include a gene which misexpress an endogenous KIAA0861 polypeptide (e.g., expression of a gene is disrupted, also known as a knockout). Such cells can serve as a model for studying disorders which are related to mutated or mis-expressed KIAA0861 alleles or for use in drug screening. Also provided are human cells (e.g., a hematopoietic stem cells) transformed with a KIAA0861 nucleic acid.

Also provided are cells or a purified preparation thereof (e.g., human cells) in which an endogenous KIAA0861 nucleic acid is under the control of a regulatory sequence that does not normally control the expression of the endogenous KIAA0861 gene. The expression characteristics of an endogenous gene within a cell (e.g., a cell line or microorganism) can be modified by inserting a heterologous DNA regulatory element into the genome of the cell such that the inserted regulatory element is operably linked to the endogenous KIAA0861 gene. For example, an endogenous KIAA0861 gene (e.g., a gene which is “transcriptionally silent,” not normally expressed, or expressed only at very low levels) may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell. Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g., Chappel, U.S. Pat. No. 5,272,071; WO 91/06667, published on May 16, 1991.

Transgenic Animals

Non-human transgenic animals that express a heterologous KIAA0861 polypeptide (e.g., expressed from a KIAA0861 nucleic acid isolated from another organism) can be generated. Such animals are useful for studying the function and/or activity of a KIAA0861 polypeptide and for identifying and/or evaluating modulators of KIAA0861 nucleic acid and KIAA0861 polypeptide activity. As used herein, a “transgenic animal” is a non-human animal such as a mammal (e.g., a non-human primate such as chimpanzee, baboon, or macaque; an ungulate such as an equine, bovine, or caprine; or a rodent such as a rat, a mouse, or an Israeli sand rat), a bird (e.g., a chicken or a turkey), an amphibian (e.g., a frog, salamander, or newt), or an insect (e.g., Drosophila melanogaster), in which one or more of the cells of the animal includes a KIAA0861 transgene. A transgene is exogenous DNA or a rearrangement (e.g., a deletion of endogenous chromosomal DNA) that is often integrated into or occurs in the genome of cells in a transgenic animal. A transgene can direct expression of an encoded gene product in one or more cell types or tissues of the transgenic animal, and other transgenes can reduce expression (e.g., a knockout). Thus, a transgenic animal can be one in which an endogenous KIAA0861 gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal (e.g., an embryonic cell of the animal) prior to development of the animal.

Intronic sequences and polyadenylation signals can also be included in the transgene to increase expression efficiency of the transgene. One or more tissue-specific regulatory sequences can be operably linked to a KIAA0861 transgene to direct expression of a KIAA0861 polypeptide to particular cells. A transgenic founder animal can be identified based upon the presence of a KIAA0861 transgene in its genome and/or expression of KIAA0861 mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a KIAA0861 polypeptide can further be bred to other transgenic animals carrying other transgenes.

KIAA0861 polypeptides can be expressed in transgenic animals or plants by introducing, for example, a nucleic acid encoding the polypeptide into the genome of an animal. In certain embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Also included is a population of cells from a transgenic animal.

KIAA0861 Polypeptides

Featured herein are isolated KIAA0861 polypeptides, which include polypeptides having amino acid sequences of SEQ ID NO: 4 or 5, and substantially identical polypeptides thereof. Such polypeptides sometimes are proteins or peptides. The polypeptide having the amino acid sequence of SEQ ID NO: 5 often is utilized, as well as domain fragments, such as a fragment containing the DH and PH domains. A KIAA0861 polypeptide is a polypeptide encoded by a KIAA0861 nucleic acid, where one nucleic acid can encode one or more different polypeptides. An “isolated” or “purified” polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. In one embodiment, the language “substantially free” means preparation of a KIAA0861 polypeptide or KIAA0861 polypeptide variant having less than about 30%, 20%, 10% and sometimes 5% (by dry weight), of non-KIAA0861 polypeptide (also referred to herein as a “contaminating protein”), or of chemical precursors or non-KIAA0861 chemicals. When the KIAA0861 polypeptide or a biologically active portion thereof is recombinantly produced, it is also often substantially free of culture medium, specifically, where culture medium represents less than about 20%, sometimes less than about 10%, and often less than about 5% of the volume of the polypeptide preparation. Isolated or purified KIAA0861 polypeptide preparations are sometimes 0.01 milligrams or more or 0.1 milligrams or more, and often 1.0 milligrams or more and 10 milligrams or more in dry weight. In specific embodiments, the KIAA0861 polypeptide comprises a leucine at amino acid position 359 in SEQ ID NO: 5, a leucine at amino acid position 378 in SEQ ID NO: 5, or an alanine at amino acid position 857 in SEQ ID NO: 5.

In another aspect, featured herein are KIAA0861 polypeptides and biologically active or antigenic fragments thereof that are useful as reagents or targets in assays applicable to prevention, treatment or diagnosis of breast cancer. In another embodiment, provided herein are KIAA0861 polypeptides having a KIAA0861 activity or activities (e.g., GTPase binding activity, guanine nucleotide exchange activity (i.e., the ability to catalyze GDP-GTP exchange reactions of Rho proteins), translocating the GEF to the plasma membrane activity (i.e., cellular localization via interactions with lipids or proteins), or recognizing the substrate GTPase activity). In certain embodiments, the polypeptides are KIAA0861 proteins including a Sec 14p-like lipid binding domain, at least one spectrin repeat (SPEC), a RhoGEF domain (or DH domain), and a Pleckstrin-homology domain (PH domain), and sometimes having a KIAA0861 activity as described herein. These domains are always found in tandem, with the PH domain found C-terminal to the DH domain. It is believed that the DH domain interacts directly with Rho GTPase depleted of GTP and Mg²⁺ while the PH domain is responsible for translocating the GEF to the plasma membrane, placing it in close proximity to the substrate GTPase. A second, or alternative role for the PH domain has recently been described and involves the direct interaction of the PH domain with the GTPase (Rossman, K L, et al. (2002) EMBO, 21 (6): 1315-1326). Methods for monitoring and quantifying this biological activity, both in vitro and in vivo, are known (see, e.g., Cheng, L, et al. (2002) MCB. 22 (19):6895-6905).

A catalytically active form of the KIAA0861 protein includes the RhoGEF domain (DH domain), which serves to catalyze GDP-GTP exchange reactions of Rho proteins, and the Pleckstrin-homology domain (PH domain), which serves to translocate the GEF to the plasma membrane. The catalytically active form of the KIAA0861 protein can be approximately 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, or 341 amino acid residues in length (from about amino acid 535, 536, 537, 538, 539, 540, 571, 572, 573, 574, 575 or 576 to amino acid 870, 871, 872, 873, 874, 875 or 876 of SEQ ID NO: 5).

Human KIAA0861 contains the following regions or other structural features: a Sec14p-like lipid binding domain at about amino acids 99 to 190 of SEQ ID NO: 5; Spectrin repeats located at about amino acid residues 333 to 503 of SEQ ID NO: 5; RhoGEF (or DH) domain at about amino acids 623 to 820 or 659 to 818 of SEQ ID NO: 5; and a Pleckstrin-homology domain (PH domain) at about amino acids 857-953 or 857-956 of SEQ ID NO: 5. DH-PH domains often span from amino acids 623-953 or 623-856 in SEQ ID NO: 5. A nucleotide sequence of a DBS gene, another guanine nucleotide exchange factor discussed hereafter, is deposited as NP_(—)079255 in the GenBank database and DB-PH regions corresponding to the KIAA0861 DB-PH region are apparent from alignments shown hereafter.

In other embodiments, there are provided methods of decreasing the guanine nucleotide exchange reactions of Rho proteins, comprising providing or administering to individuals in need of decreasing the guanine nucleotide exchange reactions of Rho proteins the pharmaceutical or physiologically acceptable composition comprising inactive human KIAA0861 protein or fragment thereof, where the inactive KIAA0861 polypeptide fragments may have introduced point mutations in the DH domain of KIAA0861 to selectively narrow its specificity of exchange, further wherein it is understood that the inactive form of KIAA0861 does not have the ability or has a decreased ability to catalyze the guanine nucleotide exchange reactions of Rho proteins, but can still bind to Rho proteins. (See “Therapeutic Treatments” Section herein).

Further included herein are KIAA0861 polypeptide fragments. The polypeptide fragment may be a domain or part of a domain of a KIAA0861 polypeptide. The polypeptide fragment is often 50 or fewer, 100 or fewer, or 200 or fewer amino acids in length, and is sometimes 300, 400, 500, 600, 700, or 900 or fewer amino acids in length. In certain embodiments, the polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids and not more than 1211 consecutive amino acids of SEQ ID NO: 5, or the polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids and not more than 543 consecutive amino acids of SEQ ID NO: 5.

KIAA0861 polypeptides described herein can be used as immunogens to produce anti-KIAA0861 antibodies in a subject, to purify KIAA0861 ligands or binding partners, and in screening assays to identify molecules which inhibit or enhance the interaction of KIAA0861 with a KIAA0861 substrate. In a preferred embodiment, KIAA0861 polypeptides described herein are used to screen for competitive inhibitors of KIAA0861 with Rho family GTP-binding proteins. Full-length KIAA0861 polypeptides and polynucleotides encoding the same may be specifically substituted for a KIAA0861 polypeptide fragment or polynucleotide encoding the same in any embodiment described herein.

Substantially identical polypeptides may depart from the amino acid sequences of SEQ ID NO: 4 or 5 in different manners. For example, conservative amino acid modifications may be introduced at one or more positions in the amino acid sequences of SEQ ID NO: 4 or 5. A “conservative amino acid substitution” is one in which the amino acid is replaced by another amino acid having a similar structure and/or chemical function. Families of amino acid residues having similar structures and functions are well known. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Also, essential and non-essential amino acids may be replaced. A “non-essential” amino acid is one that can be altered without abolishing or substantially altering the biological function of a KIAA0861 polypeptide, whereas altering an “essential” amino acid abolishes or substantially alters the biological function of a KIAA0861 polypeptide. Amino acids that are conserved among KIAA0861 polypeptides are typically essential amino acids.

Also, KIAA0861 polypeptides and polypeptide variants may exist as chimeric or fusion polypeptides. As used herein, a KIAA0861 “chimeric polypeptide” or “fusion polypeptide” includes a KIAA0861 polypeptide linked to a non-KIAA0861 polypeptide. A “non-KIAA0861 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a polypeptide which is not substantially identical to the KIAA0861 polypeptide, which includes, for example, a polypeptide that is different from the KIAA0861 polypeptide and derived from the same or a different organism. The KIAA0861 polypeptide in the fusion polypeptide can correspond to an entire or nearly entire KIAA0861 polypeptide or a fragment thereof. The non-KIAA0861 polypeptide can be fused to the N-terminus or C-terminus of the KIAA0861 polypeptide.

Fusion polypeptides can include a moiety having high affinity for a ligand. For example, the fusion polypeptide can be a GST-KIAA0861 fusion polypeptide in which the KIAA0861 sequences are fused to the C-terminus of the GST sequences, or a polyhistidine-KIAA0861 fusion polypeptide in which the KIAA0861 polypeptide is fused at the N- or C-terminus to a string of histidine residues. Such fusion polypeptides can facilitate purification of recombinant KIAA0861. Expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide), and a KIAA0861 nucleic acid can be cloned into an expression vector such that the fusion moiety is linked in-frame to the KIAA0861 polypeptide. Further, the fusion polypeptide can be a KIAA0861 polypeptide containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression, secretion, cellular internalization, and cellular localization of a KIAA0861 polypeptide can be increased through use of a heterologous signal sequence. Fusion polypeptides can also include all or a part of a serum polypeptide (e.g., an IgG constant region or human serum albumin).

KIAA0861 polypeptides or fragments thereof can be incorporated into pharmaceutical compositions and administered to a subject in vivo. Administration of these KIAA0861 polypeptides can be used to affect the bioavailability of a KIAA0861 substrate and may effectively increase or decrease KIAA0861 biological activity in a cell or effectively supplement dysfunctional or hyperactive KIAA0861 polypeptide. KIAA0861 fusion polypeptides may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a KIAA0861 polypeptide; (ii) mis-regulation of the KIAA0861 gene; and (iii) aberrant post-translational modification of a KIAA0861 polypeptide. Also, KIAA0861 polypeptides can be used as immunogens to produce anti-KIAA0861 antibodies in a subject, to purify KIAA0861 ligands or binding partners, and in screening assays to identify molecules which inhibit or enhance the interaction of KIAA0861 with a KIAA0861 substrate. Preferably, said KIAA0861 polypeptides are used in screening assays to identify molecules which inhibit the interaction of KIAA0861 with Rho family GTP-binding proteins.

In addition, polypeptides can be chemically synthesized using techniques known in the art (See, e.g., Creighton, 1983 Proteins. New York, N.Y.: W. H. Freeman and Company; and Hunkapiller et al., (1984) Nature July 12-18; 310(5973):105-11). For example, a relative short polypeptide fragment can be synthesized by use of a peptide synthesizer. Furthermore, if desired, non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the fragment sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

Also included are polypeptide fragments which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, and the like. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; and the like.

Additional post-translational modifications include, for example, N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The polypeptide fragments may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the polypeptide.

Also provided are chemically modified polypeptide derivatives that may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity. See U.S. Pat. No. 4,179,337. The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should be attached to the polypeptide with consideration of effects on functional or antigenic domains of the polypeptide. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al (1992) Exp Hematol. September; 20(8): 1028-35, reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. A polymer sometimes is attached at an amino group, such as attachment at the N-terminus or lysine group.

One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, and the like), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus may be accomplished by reductive alkylation, which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

Substantially Identical Nucleic Acids and Polypeptides

Nucleotide sequences and polypeptide sequences that are substantially identical to a KIAA0861 nucleotide sequence and the KIAA0861 polypeptide sequences encoded by those nucleotide sequences are included herein. The term “substantially identical” as used herein refers to two or more nucleic acids or polypeptides sharing one or more identical nucleotide sequences or polypeptide sequences, respectively. Included are nucleotide sequences or polypeptide sequences that are 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more (each often within a 1%, 2%, 3% or 4% variability) or more identical to the nucleotide sequences in SEQ ID NO: 1, 2 or 3 or the encoded KIAA0861 polypeptide amino acid sequences. One test for determining whether two nucleic acids are substantially identical is to determine the percent of identical nucleotide sequences or polypeptide sequences shared between the nucleic acids or polypeptides.

Calculations of sequence identity are often performed as follows. Sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is sometimes 30% or more, 40% or more, 50% or more, often 60% or more, and more often 70% or more, 80% or more, 90% or more, 90% or more, or 100% of the length of the reference sequence. The nucleotides or amino acids at corresponding nucleotide or polypeptide positions, respectively, are then compared among the two sequences. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, the nucleotides or amino acids are deemed to be identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, introduced for optimal alignment of the two sequences.

Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. Percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers & Miller, CABIOS 4: 11-17 (1989), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Also, percent identity between two amino acid sequences can be determined using the Needleman & Wunsch, J. Mol. Biol. 48: 444-453 (1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at the http address www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http address www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A set of parameters often used is a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

Another manner for determining if two nucleic acids are substantially identical is to assess whether a polynucleotide homologous to one nucleic acid will hybridize to the other nucleic acid under stringent conditions. As use herein, the term “stringent conditions” refers to conditions for hybridization and washing. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6 (1989). Aqueous and non-aqueous methods are described in that reference and either can be used. An example of stringent hybridization conditions is hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50° C. Another example of stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 55° C. A further example of stringent hybridization conditions is hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C. Often, stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C. More often, stringency conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C.

An example of a substantially identical nucleotide sequence to a KIAA0861 nucleotide sequence is one that has a different nucleotide sequence but still encodes the same polypeptide sequence encoded by the KIAA0861 nucleotide sequence. Another example is a nucleotide sequence that encodes a polypeptide having a polypeptide sequence that is more than 70% or more identical to, sometimes 75% or more, 80% or more, or 85% or more identical to, and often 90% or more and 95% or more identical to a polypeptide sequence encoded by a KIAA0861 nucleotide sequence.

KIAA0861 nucleotide sequences and KIAA0861 amino acid sequences can be used as “query sequences” to perform a search against public databases to identify other family members or related sequences, for example. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. 215: 403-10 (1990). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleotide sequences from SEQ ID NO: 1. BLAST polypeptide searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to polypeptides encoded by a KIAA0861 nucleotide sequence. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17): 3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see the http address www.ncbi.nlm.nih.gov).

A nucleic acid that is substantially identical to a KIAA0861 nucleotide sequence may include polymorphic sites at positions equivalent to those described herein when the sequences are aligned. For example, using the alignment procedures described herein, SNPs in a sequence substantially identical to a sequence in SEQ ID NO: 1, 2, or 3 can be identified at nucleotide positions that match (i.e., align) with nucleotides at SNP positions in the nucleotide sequence of SEQ ID NO: 1, 2 or 3. Also, where a polymorphic variation results in an insertion or deletion, insertion or deletion of a nucleotide sequence from a reference sequence can change the relative positions of other polymorphic sites in the nucleotide sequence.

Substantially identical nucleotide and polypeptide sequences include those that are naturally occurring, such as allelic variants (same locus), splice variants, homologs (different locus), and orthologs (different organism) or can be non-naturally occurring. Non-naturally occurring variants can be generated by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product). Orthologs, homologs, allelic variants, and splice variants can be identified using methods known in the art. These variants normally comprise a nucleotide sequence encoding a polypeptide that is 50% or more, about 55% or more, often about 70-75% or more, more often about 80-85% or more, and typically about 90-95% or more identical to the amino acid sequences of target polypeptides or a fragment thereof. Such nucleic acid molecules readily can be identified as being able to hybridize under stringent conditions to a nucleotide sequence in SEQ ID NO: 1, 2 or 3 or a fragment thereof. Nucleic acid molecules corresponding to orthologs, homologs, and allelic variants of a nucleotide sequence in SEQ ID NO: 1, 2 or 3 can be identified by mapping the sequence to the same chromosome or locus as the nucleotide sequence in SEQ ID NO: 1, 2 or 3.

Also, substantially identical nucleotide sequences may include codons that are altered with respect to the naturally occurring sequence for enhancing expression of a target polypeptide in a particular expression system. For example, the nucleic acid can be one in which one or more codons are altered, and often 10% or more or 20% or more of the codons are altered for optimized expression in bacteria (e.g., E. coli.), yeast (e.g., S. cervesiae), human (e.g., 293 cells), insect, or rodent (e.g., hamster) cells.

Methods for Identifying Subjects at Risk of Breast Cancer and Breast Cancer Risk in a Subject

Methods for prognosing and diagnosing breast cancer in subjects are provided herein. These methods include detecting the presence or absence of one or more polymorphic variations associated with breast cancer in a nucleotide sequence set forth in SEQ ID NO: 1, or substantially identical sequence thereof, in a sample from a subject, where the presence of a polymorphic variant described herein is indicative of a risk of breast cancer.

Thus, featured herein is a method for detecting a subject at risk of breast cancer or the risk of breast cancer in a subject, which comprises detecting the presence or absence of a polymorphic variation associated with breast cancer at a polymorphic site in a nucleotide sequence set forth in SEQ ID NO: 1 in a nucleic acid sample from a subject, where the nucleotide sequence comprises a polynucleotide sequence selected from the group consisting of: (a) a nucleotide sequence set forth in SEQ ID NO: 1; (b) a nucleotide sequence which encodes a polypeptide having an amino acid sequence encoded by a nucleotide sequence in SEQ ID NO: 1; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence in SEQ ID NO: 1 or a nucleotide sequence about 90% or more identical to the nucleotide sequence set forth in SEQ ID NO: 1; and (d) a fragment of a nucleotide sequence of (a), (b), or (c), often a fragment that includes a polymorphic site associated with breast cancer; whereby the presence of the polymorphic variation is indicative of a risk of breast cancer in the subject. In specific embodiments, the polymorphic variant is detected at a position corresponding to a position selected from the group consisting of rs3811728, rs3811729, rs602646, rs488277, rs1629673, rs670232, rs575326, rs575386, rs684846, rs471365, rs496251, rs831246, rs831247, rs512071, rs1502761, rs681516, rs683302, rs619424, rs620722, rs529055, rs664010, rs678454, rs2653845, rs472795, rs507079, rs534333, rs535298, rs536213, rs831245, rs639690, rs684174, rs571761, rs1983421, rs4630966, rs2314415, rs6788196, rs2103062, rs9827084, rs9864865, rs6804951, rs6770548, rs1403452, rs7609994, rs9838250, rs9863404, rs903950, rs6787284, rs2017340, rs2001449, rs1317288, rs7635891, rs10704581, rs11371910, rs10937118, rs7642053, rs3821522, rs2029926, rs1390831, rs7643890, rs11925606, rs9826325, rs6800429, rs6803368, rs1353566, rs2272115, rs2272116, rs3732603, rs940055, rs2314730, rs2030578, rs2049280, rs3732602, rs2293203, rs7639705, and position 13507 of SEQ ID NO: 1. In certain embodiments, determining the presence of a combination of two or more polymorphic variants associated with breast cancer in one or more nucleotide sequences of the sample (e.g., at ICAM, MAPK10, NUMA1, DPF3, LOC145197 and/or GALE sequences) is determined to identify a subject at risk of breast cancer and/or risk of breast cancer.

A risk of developing aggressive forms of breast cancer likely to metastasize or invade surrounding tissues (e.g., Stage IIIA, IIIB, and IV breast cancers), and subjects at risk of developing aggressive forms of breast cancer also may be identified by the methods described herein. These methods include collecting phenotype information from subjects having breast cancer, which includes the stage of progression of the breast cancer, and performing a secondary phenotype analysis to detect the presence or absence of one or more polymorphic variations associated with a particular stage form of breast cancer. Thus, detecting the presence or absence of one or more polymorphic variations in a KIAA0861 nucleotide sequence associated with a late stage form of breast cancer often is diagnostic of an aggressive form of the cancer.

Results from prognostic tests may be combined with other test results to diagnose breast cancer. For example, prognostic results may be gathered, a patient sample may be ordered based on a determined predisposition to breast cancer, the patient sample is analyzed, and the results of the analysis may be utilized to diagnose breast cancer. Also breast cancer diagnostic methods can be developed from studies used to generate prognostic/diagnostic methods in which populations are stratified into subpopulations having different progressions of breast cancer. In another embodiment, prognostic results may be gathered; a patient's risk factors for developing breast cancer analyzed (e.g., age, race, family history, age of first menstrual cycle, age at birth of first child); and a patient sample may be ordered based on a determined predisposition to breast cancer. In an alternative embodiment, the results from predisposition analyses described herein may be combined with other test results indicative of breast cancer, which were previously, concurrently, or subsequently gathered with respect to the predisposition testing. In these embodiments, the combination of the prognostic test results with other test results can be probative of breast cancer, and the combination can be utilized as a breast cancer diagnostic. The results of any test indicative of breast cancer known in the art may be combined with the methods described herein. Examples of such tests are mammography (e.g., a more frequent and/or earlier mammography regimen may be prescribed); breast biopsy and optionally a biopsy from another tissue; breast ultrasound and optionally an ultrasound analysis of another tissue; breast magnetic resonance imaging (MRI) and optionally an MRI analysis of another tissue; electrical impedance (T-scan) analysis of breast and optionally of another tissue; ductal lavage; nuclear medicine analysis (e.g., scintimammography); BRCA1 and/or BRCA2 sequence analysis results; and thermal imaging of the breast and optionally of another tissue. Testing may be performed on tissue other than breast to diagnose the occurrence of metastasis (e.g., testing of the lymph node).

Risk of breast cancer sometimes is expressed as a probability, such as an odds ratio, percentage, or risk factor. The risk is based upon the presence or absence of one or more polymorphic variants described herein, and also may be based in part upon phenotypic traits of the individual being tested. Methods for calculating predispositions based upon patient data are well known (see, e.g., Agresti, Categorical Data Analysis, 2nd Ed. 2002. Wiley). Allelotyping and genotyping analyses may be carried out in populations other than those exemplified herein to enhance the predictive power of the prognostic method. These further analyses are executed in view of the exemplified procedures described herein, and may be based upon the same polymorphic variations or additional polymorphic variations. Risk determinations for breast cancer are useful in a variety of applications. In one embodiment, breast cancer risk determinations are used by clinicians to direct appropriate detection, preventative and treatment procedures to subjects who most require these. In another embodiment, breast cancer risk determinations are used by health insurers for preparing actuarial tables and for calculating insurance premiums.

The nucleic acid sample typically is isolated from a biological sample obtained from a subject. For example, nucleic acid can be isolated from blood, saliva, sputum, urine, cell scrapings, and biopsy tissue. The nucleic acid sample can be isolated from a biological sample using standard techniques, such as the technique described in Example 2. As used herein, the term “subject” refers primarily to humans but also refers to other mammals such as dogs, cats, and ungulates (e.g., cattle, sheep, and swine). Subjects also include avians (e.g., chickens and turkeys), reptiles, and fish (e.g., salmon), as embodiments described herein can be adapted to nucleic acid samples isolated from any of these organisms. The nucleic acid sample may be isolated from the subject and then directly utilized in a method for determining the presence of a polymorphic variant, or alternatively, the sample may be isolated and then stored (e.g., frozen) for a period of time before being subjected to analysis.

The presence or absence of a polymorphic variant is determined using one or both chromosomal complements represented in the nucleic acid sample. Determining the presence or absence of a polymorphic variant in both chromosomal complements represented in a nucleic acid sample from a subject having a copy of each chromosome is useful for determining the zygosity of an individual for the polymorphic variant (i.e., whether the individual is homozygous or heterozygous for the polymorphic variant). Any oligonucleotide-based diagnostic may be utilized to determine whether a sample includes the presence or absence of a polymorphic variant in a sample. For example, primer extension methods, ligase sequence determination methods (e.g., U.S. Pat. Nos. 5,679,524 and 5,952,174, and WO 01/27326), mismatch sequence determination methods (e.g., U.S. Pat. Nos. 5,851,770; 5,958,692; 6,110,684; and 6,183,958), microarray sequence determination methods, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism detection (SSCP) (e.g., U.S. Pat. Nos. 5,891,625 and 6,013,499), PCR-based assays (e.g., TAQMAN® PCR System (Applied Biosystems)), and nucleotide sequencing methods may be used.

Oligonucleotide extension methods typically involve providing a pair of oligonucleotide primers in a polymerase chain reaction (PCR) or in other nucleic acid amplification methods for the purpose of amplifying a region from the nucleic acid sample that comprises the polymorphic variation. One oligonucleotide primer is complementary to a region 3′ of the polymorphism and the other is complementary to a region 5′ of the polymorphism. A PCR primer pair may be used in methods disclosed in U.S. Pat. Nos. 4,683,195; 4,683,202, 4,965,188; 5,656,493; 5,998,143; 6,140,054; WO 01/27327; and WO 01/27329 for example. PCR primer pairs may also be used in any commercially available machines that perform PCR, such as any of the GENEAMP® Systems available from Applied Biosystems. Also, those of ordinary skill in the art will be able to design oligonucleotide primers based upon a nucleotide sequence set forth herein without undue experimentation using knowledge readily available in the art.

Also provided is an extension oligonucleotide that hybridizes to the amplified fragment adjacent to the polymorphic variation. As used herein, the term “adjacent” refers to the 3′ end of the extension oligonucleotide being often 1 nucleotide from the 5′ end of the polymorphic site, and sometimes 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5′ end of the polymorphic site, in the nucleic acid when the extension oligonucleotide is hybridized to the nucleic acid. The extension oligonucleotide then is extended by one or more nucleotides, and the number and/or type of nucleotides that are added to the extension oligonucleotide determine whether the polymorphic variant is present. Oligonucleotide extension methods are disclosed, for example, in U.S. Pat. Nos. 4,656,127; 4,851,331; 5,679,524; 5,834,189; 5,876,934; 5,908,755; 5,912,118; 5,976,802; 5,981,186; 6,004,744; 6,013,431; 6,017,702; 6,046,005; 6,087,095; 6,210,891; and WO 01/20039. Oligonucleotide extension methods using mass spectrometry are described, for example, in U.S. Pat. Nos. 5,547,835; 5,605,798; 5,691,141; 5,849,542; 5,869,242; 5,928,906; 6,043,031; and 6,194,144, and a method often utilized is described herein in Example 2. Multiple extension oligonucleotides may be utilized in one reaction, which is referred to herein as “multiplexing.”

A microarray can be utilized for determining whether a polymorphic variant is present or absent in a nucleic acid sample. A microarray may include any oligonucleotides described herein, and methods for making and using oligonucleotide microarrays suitable for diagnostic use are disclosed in U.S. Pat. Nos. 5,492,806; 5,525,464; 5,589,330; 5,695,940; 5,849,483; 6,018,041; 6,045,996; 6,136,541; 6,142,681; 6,156,501; 6,197,506; 6,223,127; 6,225,625; 6,229,911; 6,239,273; WO 00/52625; WO 01/25485; and WO 01/29259. The microarray typically comprises a solid support and the oligonucleotides may be linked to this solid support by covalent bonds or by non-covalent interactions. The oligonucleotides may also be linked to the solid support directly or by a spacer molecule. A microarray may comprise one or more oligonucleotides complementary to a polymorphic site shown in SEQ ID NO: 1 or below.

A kit also may be utilized for determining whether a polymorphic variant is present or absent in a nucleic acid sample. A kit often comprises one or more pairs of oligonucleotide primers useful for amplifying a fragment of a KIAA0861 nucleotide sequence or a substantially identical sequence thereof, where the fragment includes a polymorphic site. The kit sometimes comprises a polymerizing agent, for example, a thermostable nucleic acid polymerase such as one disclosed in U.S. Pat. No. 4,889,818 or 6,077,664. Also, the kit often comprises an elongation oligonucleotide that hybridizes to a KIAA0861 nucleotide sequence in a nucleic acid sample adjacent to the polymorphic site. Where the kit includes an elongation oligonucleotide, it also often comprises chain elongating nucleotides, such as DATP, dTTP, dGTP, dCTP, and dITP, including analogs of dATP, dTTP, dGTP, dCTP and dITP, provided that such analogs are substrates for a thermostable nucleic acid polymerase and can be incorporated into a nucleic acid chain elongated from the extension oligonucleotide. Along with chain elongating nucleotides would be one or more chain terminating nucleotides such as ddATP, ddTTP, ddGTP, ddCTP, and the like. In an embodiment, the kit comprises one or more oligonucleotide primer pairs, a polymerizing agent, chain elongating nucleotides, at least one elongation oligonucleotide, and one or more chain terminating nucleotides. Kits optionally include buffers, vials, microtiter plates, and instructions for use.

An individual identified as being at risk of breast cancer may be heterozygous or homozygous with respect to the allele associated with a higher risk of breast cancer. A subject homozygous for an allele associated with an increased risk of breast cancer is at a comparatively high risk of breast cancer, a subject heterozygous for an allele associated with an increased risk of breast cancer is at a comparatively intermediate risk of breast cancer, and a subject homozygous for an allele associated with a decreased risk of breast cancer is at a comparatively low risk of breast cancer. A genotype may be assessed for a complementary strand, such that the complementary nucleotide at a particular position is detected.

Also featured are methods for determining risk of breast cancer and/or identifying a subject at risk of breast cancer by contacting a polypeptide or protein encoded by a KIAA0861 nucleotide sequence from a subject with an antibody that specifically binds to an epitope associated with increased risk of breast cancer in the polypeptide. In certain embodiments, the antibody specifically binds to an epitope that comprises a leucine at amino acid position 359 in SEQ ID NO: 5, a leucine at amino acid position 378 in SEQ ID NO: 5, or an alanine at amino acid position 857 in SEQ ID NO: 5.

Applications of Prognostic and Diagnostic Results to Pharmacogenomic Methods

Pharmacogenomics is a discipline that involves tailoring a treatment for a subject according to the subject's genotype. For example, based upon the outcome of a prognostic test described herein, a clinician or physician may target pertinent information and preventative or therapeutic treatments to a subject who would be benefited by the information or treatment and avoid directing such information and treatments to a subject who would not be benefited (e.g., the treatment has no therapeutic effect and/or the subject experiences adverse side effects). As therapeutic approaches for breast cancer continue to evolve and improve, the goal of treatments for breast cancer related disorders is to intervene even before clinical signs (e.g., identification of lump in the breast) first manifest. Thus, genetic markers associated with susceptibility to breast cancer prove useful for early diagnosis, prevention and treatment of breast cancer.

The following is an example of a pharmacogenomic embodiment. A particular treatment regimen can exert a differential effect depending upon the subject's genotype. Where a candidate therapeutic exhibits a significant interaction with a major allele and a comparatively weak interaction with a minor allele (e.g., an order of magnitude or greater difference in the interaction), such a therapeutic typically would not be administered to a subject genotyped as being homozygous for the minor allele, and sometimes not administered to a subject genotyped as being heterozygous for the minor allele. In another example, where a candidate therapeutic is not significantly toxic when administered to subjects who are homozygous for a major allele but is comparatively toxic when administered to subjects heterozygous or homozygous for a minor allele, the candidate therapeutic is not typically administered to subjects who are genotyped as being heterozygous or homozygous with respect to the minor allele.

The methods described herein are applicable to pharmacogenomic methods for detecting, preventing, alleviating and/or treating breast cancer. For example, a nucleic acid sample from an individual may be subjected to a genetic test described herein. Where one or more polymorphic variations associated with increased risk of breast cancer are identified in a subject, information for detecting, preventing or treating breast cancer and/or one or more breast cancer detection, prevention and/or treatment regimens then may be directed to and/or prescribed to that subject.

In certain embodiments, a detection, prevenative and/or treatment regimen is specifically prescribed and/or administered to individuals who will most benefit from it based upon their risk of developing breast cancer assessed by the methods described herein. Thus, provided are methods for identifying a subject at risk of breast cancer and then prescribing a detection, therapeutic or preventative regimen to individuals identified as being at risk of breast cancer. Thus, certain embodiments are directed to methods for treating breast cancer in a subject, reducing risk of breast cancer in a subject, or early detection of breast cancer in a subject, which comprise: detecting the presence or absence of a polymorphic variant associated with breast cancer in a nucleotide sequence set forth in SEQ ID NO: 1 in a nucleic acid sample from a subject, where the nucleotide sequence comprises a polynucleotide sequence selected from the group consisting of: (a) a nucleotide sequence set forth in SEQ ID NO: 1; (b) a nucleotide sequence which encodes a polypeptide having an amino acid sequence encoded by a nucleotide sequence in SEQ ID NO: 1; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence in SEQ ID NO: 1 or a nucleotide sequence about 90% or more identical to the nucleotide sequence set forth in SEQ ID NO: 1; and (d) a fragment of a nucleotide sequence of (a), (b), or (c), sometimes comprising a polymorphic site associated with breast cancer; and prescribing or administering a breast cancer treatment regimen, preventative regimen and/or detection regimen to a subject from whom the sample originated where the presence of one or more polymorphic variations associated with breast cancer are detected in the nucleotide sequence. In certain embodiments, one or more of the polymorphic variants described herein is detected. In these methods, genetic results may be utilized in combination with other test results to diagnose breast cancer as described above. Other test results include but are not limited to mammography results, imaging results, biopsy results and results from BRCA1 or BRAC2 test results, as described above.

Detection regimens include one or more mammography procedures, a regular mammography regimen (e.g., once a year, or once every six, four, three or two months); an early mammography regimen (e.g., mammography tests are performed beginning at age 25, 30, or 35); one or more biopsy procedures (e.g., a regular biopsy regimen beginning at age 40); breast biopsy and biopsy from other tissue; breast ultrasound and optionally ultrasound analysis of another tissue; breast magnetic resonance imaging (MRI) and optionally MRI analysis of another tissue; electrical impedance (T-scan) analysis of breast and optionally another tissue; ductal lavage; nuclear medicine analysis (e.g., scintimammography); BRCA1 and/or BRCA2 sequence analysis results; and/or thermal imaging of the breast and optionally another tissue.

Treatments sometimes are preventative (e.g., is prescribed or administered to reduce the probability that a breast cancer associated condition arises or progresses), sometimes are therapeutic, and sometimes delay, alleviate or halt the progression of breast cancer. Any known preventative or therapeutic treatment for alleviating or preventing the occurrence of breast cancer is prescribed and/or administered. For example, certain preventative treatments often are prescribed to subjects having a predisposition to breast cancer and where the subject is not diagnosed with breast cancer or is diagnosed as having symptoms indicative of early stage breast cancer (e.g., stage I). For subjects not diagnosed as having breast cancer, any preventative treatments known in the art can be prescribed and administered, which include selective hormone receptor modulators (e.g., selective estrogen receptor modulators (SERMs) such as tamoxifen, reloxifene, and toremifene); compositions that prevent production of hormones (e.g., aramotase inhibitors that prevent the production of estrogen in the adrenal gland, such as exemestane, letrozole, anastrozol, groserelin, and megestrol); other hormonal treatments (e.g., goserelin acetate and fulvestrant); biologic response modifiers such as antibodies (e.g., trastuzumab (herceptin/HER2)); anthracycline antibiotics (e.g., ellence/Pharmorubicin®); surgery (e.g., lumpectomy and mastectomy); drugs that delay or halt metastasis (e.g., pamidronate disodium); and alternative/complementary medicine (e.g., acupuncture, acupressure, moxibustion, qi gong, reiki, ayurveda, vitamins, minerals, and herbs (e.g., astragalus root, burdock root, garlic, green tea, and licorice root)).

The use of breast cancer treatments are well known in the art, and include surgery, chemotherapy and/or radiation therapy. Any of the treatments may be used in combination to treat or prevent breast cancer (e.g., surgery followed by radiation therapy or chemotherapy). Examples of chemotherapeutics are taxanes (e.g., docetaxel or paclitaxel), and examples of chemotherapy combinations used to treat breast cancer include: cyclophosphamide (Cytoxan), methotrexate (Amethopterin, Mexate, Folex), and fluorouracil (Fluorouracil, 5-Fu, Adrucil), which is referred to as CMF; cyclophosphamide, doxorubicin (Adriamycin), and fluorouracil, which is referred to as CAF; and doxorubicin (Adriamycin) and cyclophosphamide, which is referred to as AC.

As breast cancer preventative and treatment information can be specifically targeted to subjects in need thereof (e.g., those at risk of developing breast cancer or those that have early signs of breast cancer), provided herein is a method for preventing or reducing the risk of developing breast cancer in a subject, which comprises: (a) detecting the presence or absence of a polymorphic variation associated with breast cancer at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject; (b) identifying a subject with a predisposition to breast cancer, whereby the presence of the polymorphic variation is indicative of a predisposition to breast cancer in the subject; and (c) if such a predisposition is identified, providing the subject with information about methods or products to prevent or reduce breast cancer or to delay the onset of breast cancer. Also provided is a method of targeting information or advertising to a subpopulation of a human population based on the subpopulation being genetically predisposed to a disease or condition, which comprises: (a) detecting the presence or absence of a polymorphic variation associated with breast cancer at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject; (b) identifying the subpopulation of subjects in which the polymorphic variation is associated with breast cancer; and (c) providing information only to the subpopulation of subjects about a particular product which may be obtained and consumed or applied by the subject to help prevent or delay onset of the disease or condition.

Pharmacogenomics methods also may be used to analyze and predict a response to a breast cancer treatment or a drug. For example, if pharmacogenomics analysis indicates a likelihood that an individual will respond positively to a breast cancer treatment with a particular drug, the drug may be administered to the individual. Conversely, if the analysis indicates that an individual is likely to respond negatively to treatment with a particular drug, an alternative course of treatment may be prescribed. A negative response may be defined as either the absence of an efficacious response or the presence of toxic side effects. The response to a therapeutic treatment can be predicted in a background study in which subjects in any of the following populations are genotyped: a population that responds favorably to a treatment regimen, a population that does not respond significantly to a treatment regimen, and a population that responds adversely to a treatment regiment (e.g., exhibits one or more side effects). These populations are provided as examples and other populations and subpopulations may be analyzed. Based upon the results of these analyses, a subject is genotyped to predict whether he or she will respond favorably to a treatment regimen, not respond significantly to a treatment regimen, or respond adversely to a treatment regimen.

The methods described herein also are applicable to clinical drug trials. One or more polymorphic variants indicative of response to an agent for treating breast cancer or to side effects to an agent for treating breast cancer may be identified using the methods described herein. Thereafter, potential participants in clinical trials of such an agent may be screened to identify those individuals most likely to respond favorably to the drug and exclude those likely to experience side effects. In that way, the effectiveness of drug treatment may be measured in individuals who respond positively to the drug, without lowering the measurement as a result of the inclusion of individuals who are unlikely to respond positively in the study and without risking undesirable safety problems. In certain embodiments, the agent for treating breast cancer described herein targets KIAA0861 or a target in the KIAA0861 pathway (e.g., Rho GTPase).

Thus, another embodiment is a method of selecting an individual for inclusion in a clinical trial of a treatment or drug comprising the steps of: (a) obtaining a nucleic acid sample from an individual; (b) determining the identity of a polymorphic variation which is associated with a positive response to the treatment or the drug, or at least one polymorphic variation which is associated with a negative response to the treatment or the drug in the nucleic acid sample, and (c) including the individual in the clinical trial if the nucleic acid sample contains said polymorphic variation associated with a positive response to the treatment or the drug or if the nucleic acid sample lacks said polymorphic variation associated with a negative response to the treatment or the drug. In addition, the methods for selecting an individual for inclusion in a clinical trial of a treatment or drug encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination. The polymorphic variation may be in a sequence selected individually or in any combination from the group consisting of (i) a polynucleotide sequence set forth in SEQ ID NO: 1; (ii) a polynucleotide sequence that is 90% or more identical to a nucleotide sequence set forth in SEQ ID NO: 1; (iii) a polynucleotide sequence that encodes a polypeptide having an amino acid sequence identical to or 90% or more identical to an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NO: 1; and (iv) a fragment of a polynucleotide sequence of (i), (ii), or (iii) comprising the polymorphic site. The including step (c) optionally comprises administering the drug or the treatment to the individual if the nucleic acid sample contains the polymorphic variation associated with a positive response to the treatment or the drug and the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug.

Also provided herein is a method of partnering between a diagnostic/prognostic testing provider and a provider of a consumable product, which comprises: (a) the diagnostic/prognostic testing provider detects the presence or absence of a polymorphic variation associated with breast cancer at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject; (b) the diagnostic/prognostic testing provider identifies the subpopulation of subjects in which the polymorphic variation is associated with breast cancer; (c) the diagnostic/prognostic testing provider forwards information to the subpopulation of subjects about a particular product which may be obtained and consumed or applied by the subject to help prevent or delay onset of the disease or condition; and (d) the provider of a consumable product forwards to the diagnostic test provider a fee every time the diagnostic/prognostic test provider forwards information to the subject as set forth in step (c) above.

Compositions Comprising Breast Cancer-Directed Molecules

Featured herein is a composition comprising a breast cancer cell and one or more molecules specifically directed and targeted to a nucleic acid comprising a KIAA0861 nucleotide sequence or a KIAA0861 polypeptide. Such directed molecules include, but are not limited to, a compound that binds to a KIAA0861 nucleic acid or a KIAA0861 polypeptide; a RNAi or siRNA molecule having a strand complementary to a KIAA0861 nucleotide sequence; an antisense nucleic acid complementary to an RNA encoded by a KIAA0861 DNA sequence; a ribozyme that hybridizes to a KIAA0861 nucleotide sequence; a nucleic acid aptamer that specifically binds a KIAA0861 polypeptide; and an antibody that specifically binds to a KIAA0861 polypeptide or binds to a KIAA0861 nucleic acid. In certain embodiments, the antibody specifically binds to an epitope that comprises a leucine at amino acid position 359 in SEQ ID NO: 5, a leucine at amino acid position 378 in SEQ ID NO: 5, or an alanine at amino acid position 857 in SEQ ID NO: 5. In specific embodiments, the breast cancer directed molecule interacts with a KIAA0861 nucleic acid or polypeptide variant associated with breast cancer. In other embodiments, the breast cancer directed molecule interacts with a polypeptide involved in the KIAA0861 signal pathway, or a nucleic acid encoding such a polypeptide. Polypeptides involved in the KIAA0861 signal pathway are discussed herein.

Compositions sometimes include an adjuvant known to stimulate an immune response, and in certain embodiments, an adjuvant that stimulates a T-cell lymphocyte response. Adjuvants are known, including but not limited to an aluminum adjuvant (e.g., aluminum hydroxide); a cytokine adjuvant or adjuvant that stimulates a cytokine response (e.g., interleukin (IL)-12 and/or γ-interferon cytokines); a Freund-type mineral oil adjuvant emulsion (e.g., Freund's complete or incomplete adjuvant); a synthetic lipoid compound; a copolymer adjuvant (e.g., TitreMax); a saponin; Quil A; a liposome; an oil-in-water emulsion (e.g., an emulsion stabilized by Tween 80 and pluronic polyoxyethlene/polyoxypropylene block copolymer (Syntex Adjuvant Formulation); TitreMax; detoxified endotoxin (MPL) and mycobacterial cell wall components (TDW, CWS) in 2% squalene (Ribi Adjuvant System)); a muramyl dipeptide; an immune-stimulating complex (ISCOM, e.g., an Ag-modified saponin/cholesterol micelle that forms stable cage-like structure); an aqueous phase adjuvant that does not have a depot effect (e.g., Gerbu adjuvant); a carbohydrate polymer (e.g., AdjuPrime); L-tyrosine; a manide-oleate compound (e.g., Montanide); an ethylene-vinyl acetate copolymer (e.g., Elvax 40W1,2); or lipid A, for example. Such compositions are useful for generating an immune response against a breast cancer directed molecule (e.g., an HLA-binding subsequence within a polypeptide encoded by a nucleotide sequence in SEQ ID NO: 1). In such methods, a peptide having an amino acid subsequence of a polypeptide encoded by a nucleotide sequence in SEQ ID NO: 1 is delivered to a subject, where the subsequence binds to an HLA molecule and induces a CTL lymphocyte response. The peptide sometimes is delivered to the subject as an isolated peptide or as a minigene in a plasmid that encodes the peptide. Methods for identifying HLA-binding subsequences in such polypeptides are known (see e.g., publication WO02/20616 and PCT application US98/01373 for methods of identifying such sequences).

The breast cancer cell may be in a group of breast cancer cells and/or other types of cells cultured in vitro or in a tissue having breast cancer cells (e.g., a melanocytic lesion) maintained in vitro or present in an animal in vivo (e.g., a rat, mouse, ape or human). In certain embodiments, a composition comprises a component from a breast cancer cell or from a subject having a breast cancer cell instead of the breast cancer cell or in addition to the breast cancer cell, where the component sometimes is a nucleic acid molecule (e.g., genomic DNA), a protein mixture or isolated protein, for example. The aforementioned compositions have utility in diagnostic, prognostic and pharmacogenomic methods described previously and in breast cancer therapeutics described hereafter. Certain breast cancer molecules are described in greater detail below.

Compounds

Compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive (see, e.g., Zuckermann et al, J. Med. Chem. 37: 2678-85 (1994)); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; “one-bead one-compound” library methods; and synthetic library methods using affinity chromatography selection. Biological library and peptoid library approaches are typically limited to peptide libraries, while the other approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145, (1997)). Examples of methods for synthesizing molecular libraries are described, for example, in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90: 6909 (1993); Erb et al, Proc. Natl. Acad. Sci. USA 91: 11422 (1994); Zuckermann et al., J. Med. Chem. 37: 2678 (1994); Cho et al., Science 261: 1303 (1993); Carrell et al., Angew. Chem. Int. Ed. Engl. 33: 2059 (1994); Carell et al., Angew. Chem. Int. Ed. Engl. 33: 2061 (1994); and in Gallop et al., J. Med. Chem. 37: 1233 (1994).

Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13: 412-421 (1992)), or on beads (Lam, Nature 354: 82-84 (1991)), chips (Fodor, Nature 364: 555-556 (1993)), bacteria or spores (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al., Proc. Natl. Acad. Sci. USA 89: 1865-1869 (1992)) or on phage (Scott and Smith, Science 249: 386-390 (1990); Devlin, Science 249: 404-406 (1990); Cwirla et al., Proc. Natl. Acad. Sci. 87: 6378-6382 (1990); Felici, J. Mol. Biol. 222: 301-310 (1991); Ladner supra.).

A compound sometimes alters expression and sometimes alters activity of a KIAA0861 polypeptide and may be a small molecule. Small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

Antisense Nucleic Acid Molecules, Ribozymes, RNAi, siRNA and Modified Nucleic Acid Molecules

An “antisense” nucleic acid refers to a nucleotide sequence complementary to a “sense” nucleic acid encoding a polypeptide, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire coding strand in SEQ ID NO: 1, 2 or 3, or to a portion thereof or a substantially identical sequence thereof. In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence in SEQ ID NO: 1 (e.g., 5′ and 3′ untranslated regions).

An antisense nucleic acid can be designed such that it is complementary to the entire coding region of an mRNA encoded by a nucleotide sequence in SEQ ID NO: 1 (e.g., SEQ ID NO: 2 or 3), and often the antisense nucleic acid is an oligonucleotide antisense to only a portion of a coding or noncoding region of the mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the mRNA, e.g., between the −10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length. The antisense nucleic acids, which include the ribozymes described hereafter, can be designed to target a nucleotide sequence in SEQ ID NO: 1, often a variant associated with breast cancer, or a substantially identical sequence thereof. Among the variants, minor alleles and major alleles can be targeted, and those associated with a higher risk of breast cancer are often designed, tested, and administered to subjects.

An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using standard procedures. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

When utilized as therapeutics, antisense nucleic acids typically are administered to a subject (e.g., by direct injection at a tissue site) or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a polypeptide and thereby inhibit expression of the polypeptide, for example, by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then are administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, for example, by linking antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. Sufficient intracellular concentrations of antisense molecules are achieved by incorporating a strong promoter, such as a pol II or pol III promoter, in the vector construct.

Antisense nucleic acid molecules sometimes are alpha-anomeric nucleic acid molecules. An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual beta-units, the strands run parallel to each other (Gaultier et al., Nucleic Acids. Res. 15: 6625-6641 (1987)). Antisense nucleic acid molecules can also comprise a 2′-o-methylribonucleotide (Inoue et al., Nucleic Acids Res. 15: 6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215: 327-330 (1987)). Antisense nucleic acids sometimes are composed of DNA or PNA or any other nucleic acid derivatives described previously.

In another embodiment, an antisense nucleic acid is a ribozyme. A ribozyme having specificity for a KIAA0861 nucleotide sequence can include one or more sequences complementary to such a nucleotide sequence, and a sequence having a known catalytic region responsible for mRNA cleavage (see e.g., U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach, Nature 334: 585-591 (1988)). For example, a derivative of a Tetrahymena L-19 IVS RNA is sometimes utilized in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a mRNA (see e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742). Also, target mRNA sequences can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see e.g., Bartel & Szostak, Science 261: 1411-1418 (1993)).

Breast cancer directed molecules include in certain embodiments nucleic acids that can form triple helix structures with a KIAA0861 nucleotide sequence or a substantially identical sequence thereof, especially one that includes a regulatory region that controls expression of a polypeptide. Gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of a KIAA0861 nucleotide sequence or a substantially identical sequence (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of a gene in target cells (see e.g., Helene, Anticancer Drug Des. 6(6): 569-84 (1991); Helene et al., Ann. N.Y. Acad. Sci. 660: 27-36 (1992); and Maher, Bioassays 14(12): 807-15 (1992). Potential sequences that can be targeted for triple helix formation can be increased by creating a so-called “switchback” nucleic acid molecule. Switchback molecules are synthesized in an alternating 5′-3′,3′-5′ manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

Breast cancer directed molecules include RNAi and siRNA nucleic acids. Gene expression may be inhibited by the introduction of double-stranded RNA (dsRNA), which induces potent and specific gene silencing, a phenomenon called RNA interference or RNAi. See, e.g., Fire et al., U.S. Pat. No. 6,506,559; Tuschl et al. PCT International Publication No. WO 01/75164; Kay et al. PCT International Publication No. WO 03/010180A1; or Bosher J M, Labouesse, Nat Cell Biol 2000 February; 2(2):E31-6. This process has been improved by decreasing the size of the double-stranded RNA to 20-24 base pairs (to create small-interfering RNAs or siRNAs) that “switched off” genes in mammalian cells without initiating an acute phase response, i.e., a host defense mechanism that often results in cell death (see, e.g., Caplen et al. Proc Natl Acad Sci USA. 2001 Aug. 14; 98(17):9742-7 and Elbashir et al. Methods 2002 February; 26(2):199-213). There is increasing evidence of post-transcriptional gene silencing by RNA interference (RNAi) for inhibiting targeted expression in mammalian cells at the mRNA level, in human cells. There is additional evidence of effective methods for inhibiting the proliferation and migration of tumor cells in human patients, and for inhibiting metastatic cancer development (see, e.g., U.S. Patent Application No. US2001000993183; Caplen et al. Proc Natl Acad Sci USA; and Abderrahmani et al. Mol Cell Biol 2001 Nov. 21 (21):7256-67).

An “siRNA” or “RNAi” refers to a nucleic acid that forms a double stranded RNA and has the ability to reduce or inhibit expression of a gene or target gene when the siRNA is delivered to or expressed in the same cell as the gene or target gene. “siRNA” refers to short double-stranded RNA formed by the complementary strands. Complementary portions of the siRNA that hybridize to form the double stranded molecule often have substantial or complete identity to the target molecule sequence. In one embodiment, an siRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded siRNA.

When designing the siRNA molecules, the targeted region often is selected from a given DNA sequence beginning 50 to 100 nucleotides downstream of the start codon. See, e.g., Elbashir et al., Methods 26:199-213 (2002). Initially, 5′ or 3′ UTRs and regions nearby the start codon were avoided assuming that UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP or RISC endonuclease complex. Sometimes regions of the target 23 nucleotides in length conforming to the sequence motif AA(N19)TT (SEQ ID NO: 6) (N, an nucleotide), and regions with approximately 30% to 70% G/C-content (often about 50% G/C-content) often are selected. If no suitable sequences are found, the search often is extended using the motif NA(N21). The sequence of the sense siRNA sometimes corresponds to (N19) TT or N21 (position 3 to 23 of the 23-nt motif), respectively. In the latter case, the 3′ end of the sense siRNA often is converted to TT. The rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense 3′ overhangs. The antisense siRNA is synthesized as the complement to position 1 to 21 of the 23-nt motif. Because position 1 of the 23-nt motif is not recognized sequence-specifically by the antisense siRNA, the 3′-most nucleotide residue of the antisense siRNA can be chosen deliberately. However, the penultimate nucleotide of the antisense siRNA (complementary to position 2 of the 23-nt motif) often is complementary to the targeted sequence. For simplifying chemical synthesis, TT often is utilized. siRNAs corresponding to the target motif NAR(N17)YNN, where R is purine (A,G) and Y is pyrimidine (C,U), often are selected. Respective 21 nucleotide sense and antisense siRNAs often begin with a purine nucleotide and can also be expressed from pol III expression vectors without a change in targeting site. Expression of RNAs from pol III promoters often is efficient when the first transcribed nucleotide is a purine.

The sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof. Often, the siRNA is about 15 to about 50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, sometimes about 20-30 nucleotides in length or about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The siRNA sometimes is about 21 nucleotides in length. Methods of using siRNA are well known in the art, and specific siRNA molecules may be purchased from a number of companies including Dharmacon Research, Inc.

Antisense, ribozyme, RNAi and siRNA nucleic acids can be altered to form modified nucleic acid molecules. The nucleic acids can be altered at base moieties, sugar moieties or phosphate backbone moieties to improve stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup et al., Bioorganic & Medicinal Chemistry 4 (1): 5-23 (1996)). As used herein, the terms “peptide nucleic acid” or “PNA” refers to a nucleic acid mimic such as a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. Synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described, for example, in Hyrup et al, (1996) supra and Perry-O'Keefe et al, Proc. Natl. Acad. Sci. 93: 14670-675 (1996).

PNA nucleic acids can be used in prognostic, diagnostic, and therapeutic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNA nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as “artificial restriction enzymes” when used in combination with other enzymes, (e.g., S1 nucleases (Hyrup (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup et al, (1996) supra; Perry-O'Keefe supra).

In other embodiments, oligonucleotides may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across cell membranes (see e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA 86: 6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci. USA 84: 648-652 (1987); PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al., Bio-Techniques 6: 958-976 (1988)) or intercalating agents. (See, e.g., Zon, Pharm. Res. 5: 539-549 (1988)). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

Also included herein are molecular beacon oligonucleotide primer and probe molecules having one or more regions complementary to a nucleotide sequence of SEQ ID NO: 1, 2, or 3 or a substantially identical sequence thereof, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantifying the presence of the nucleic acid in a sample. Molecular beacon nucleic acids are described, for example, in Lizardi et al., U.S. Pat. No. 5,854,033; Nazarenko et al., U.S. Pat. No. 5,866,336, and Livak et al., U.S. Pat. No. 5,876,930.

Antibodies

The term “antibody” as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab′)₂ fragments which can be generated by treating the antibody with an enzyme such as pepsin. An antibody sometimes is a polyclonal, monoclonal, recombinant (e.g., a chimeric or humanized), fully human, non-human (e.g., murine), or a single chain antibody. An antibody may have effector function and can fix complement, and is sometimes coupled to a toxin or imaging agent.

A full-length polypeptide or antigenic peptide fragment encoded by a KIAA0861 nucleotide sequence can be used as an immunogen or can be used to identify antibodies made with other immunogens, e.g., cells, membrane preparations, and the like. An antigenic peptide often includes at least 8 amino acid residues of the amino acid sequences encoded by a nucleotide sequence of SEQ ID NO: 1, 2 or 3, or substantially identical sequence thereof, and encompasses an epitope. Antigenic peptides sometimes include 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, or 30 or more amino acids. Hydrophilic and hydrophobic fragments of polypeptides sometimes are used as immunogens.

Epitopes encompassed by the antigenic peptide are regions located on the surface of the polypeptide (e.g., hydrophilic regions) as well as regions with high antigenicity. For example, an Emini surface probability analysis of the human polypeptide sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface of the polypeptide and are thus likely to constitute surface residues useful for targeting antibody production. The antibody may bind an epitope on any domain or region on polypeptides described herein.

Also, chimeric, humanized, and completely human antibodies are useful for applications which include repeated administration to subjects. Chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, can be made using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al International Application No. PCT/US86/02269; Akira, et al European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al European Patent Application 173,494; Neuberger et al PCT International Publication No. WO 86/01533; Cabilly et al U.S. Pat. No. 4,816,567; Cabilly et al European Patent Application 125,023; Better et al, Science 240: 1041-1043 (1988); Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443 (1987); Liu et al., J. Immunol. 139: 3521-3526 (1987); Sun et al., Proc. Natl. Acad. Sci. USA 84: 214-218 (1987); Nishimura et al., Canc. Res. 47: 999-1005 (1987); Wood et al., Nature 314: 446-449 (1985); and Shaw et al., J. Natl. Cancer Inst. 80: 1553-1559 (1988); Morrison, S. L., Science 229: 1202-1207 (1985); Oi et al., BioTechniques 4: 214 (1986); Winter U.S. Pat. No. 5,225,539; Jones et al., Nature 321: 552-525 (1986); Verhoeyan et al., Science 239: 1534; and Beidler et al., J. Immunol. 141: 4053-4060 (1988).

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. See, for example, Lonberg and Huszar, Int. Rev. Immunol. 13: 65-93 (1995); and U.S. Pat. Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806. In addition, companies such as Abgenix, Inc. (Fremont, Calif.) and Medarex, Inc. (Princeton, N.J.), can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above. Completely human antibodies that recognize a selected epitope also can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody (e.g., a murine antibody) is used to guide the selection of a completely human antibody recognizing the same epitope. This technology is described for example by Jespers et al., Bio/Technology 12: 899-903 (1994).

Antibody can be a single chain antibody. A single chain antibody (scFV) can be engineered (see, e.g., Colcher et al., Ann. N Y Acad. Sci. 880: 263-80 (1999); and Reiter, Clin. Cancer Res. 2: 245-52 (1996)). Single chain antibodies can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target polypeptide.

Antibodies also may be selected or modified so that they exhibit reduced or no ability to bind an Fc receptor. For example, an antibody may be an isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor (e.g., it has a mutagenized or deleted Fc receptor binding region).

Also, an antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

Antibody conjugates can be used for modifying a given biological response. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a polypeptide such as tumor necrosis factor, γ-interferon, α-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors. Also, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, for example.

An antibody (e.g., monoclonal antibody) can be used to isolate target polypeptides by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an antibody can be used to detect a target polypeptide (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide. Antibodies can be used diagnostically to monitor polypeptide levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labeling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H. Also, an antibody can be utilized as a test molecule for determining whether it can treat breast cancer, and as a therapeutic for administration to a subject for treating breast cancer.

An antibody can be made by immunizing with a purified antigen, or a fragment thereof, e.g., a fragment described herein, a membrane associated antigen, tissues, e.g., crude tissue preparations, whole cells, preferably living cells, lysed cells, or cell fractions.

Included herein are antibodies which bind only a native polypeptide, only denatured or otherwise non-native polypeptide, or which bind both, as well as those having linear or conformational epitopes. Conformational epitopes sometimes can be identified by selecting antibodies that bind to native but not denatured polypeptide. Also featured are antibodies that specifically bind to a polypeptide variant associated with breast cancer.

Screening Assays

Featured herein are methods for identifying a candidate therapeutic for treating breast cancer. The methods comprise contacting a test molecule with a target molecule in a system. A “target molecule” as used herein refers to a nucleic acid of SEQ ID NO: 1, 2 or 3, a substantially identical nucleic acid thereof, or a fragment thereof, and an encoded polypeptide of the foregoing. The method also comprises determining the presence or absence of an interaction between the test molecule and the target molecule, where the presence of an interaction between the test molecule and the nucleic acid or polypeptide identifies the test molecule as a candidate breast cancer therapeutic. The interaction between the test molecule and the target molecule may be quantified.

Test molecules and candidate therapeutics include, but are not limited to, compounds, antisense nucleic acids, siRNA molecules, ribozymes, polypeptides or proteins encoded by a KIAA0861 nucleic acids, or a substantially identical sequence or fragment thereof, and immunotherapeutics (e.g., antibodies and HLA-presented polypeptide fragments). A test molecule or candidate therapeutic may act as a modulator of target molecule concentration or target molecule function in a system. A “modulator” may agonize (i.e., up-regulates) or antagonize (i.e., down-regulates) a target molecule concentration partially or completely in a system by affecting such cellular functions as DNA replication and/or DNA processing (e.g., DNA methylation or DNA repair), RNA transcription and/or RNA processing (e.g., removal of intronic sequences and/or translocation of spliced mRNA from the nucleus), polypeptide production (e.g., translation of the polypeptide from mRNA), and/or polypeptide post-translational modification (e.g., glycosylation, phosphorylation, and proteolysis of pro-polypeptides). A modulator may also agonize or antagonize a biological function of a target molecule partially or completely, where the function may include adopting a certain structural conformation, interacting with one or more binding partners, ligand binding, catalysis (e.g., phosphorylation, dephosphorylation, hydrolysis, methylation, and isomerization), and an effect upon a cellular event (e.g., effecting progression of breast cancer).

As used herein, the term “system” refers to a cell free in vitro environment and a cell-based environment such as a collection of cells, a tissue, an organ, or an organism. A system is “contacted” with a test molecule in a variety of manners, including adding molecules in solution and allowing them to interact with one another by diffusion, cell injection, and any administration routes in an animal. As used herein, the term “interaction” refers to an effect of a test molecule on test molecule, where the effect sometimes is binding between the test molecule and the target molecule, and sometimes is an observable change in cells, tissue, or organism.

There are many standard methods for detecting the presence or absence of an interaction between a test molecule and a target molecule. For example, titrametric, acidimetric, radiometric, NMR, monolayer, polarographic, spectrophotometric, fluorescent, and ESR assays probative of a target molecule interaction may be utilized.

KIAA0861 activity and/or KIAA0861 interactions can be detected and quantified using assays known in the art. For example, an immunoprecipitation assay or a kinase activity assay that employs a kinase-inactivated MEK can be utilized. Kinase inactivated MEKs are known in the art, such as a MEK that includes the mutation K97M. In these assays, mammalian cells (e.g., COS or NIH-3T3) are transiently transfected with constructs expressing KIAA0861, and in addition, the cells are co-transfected with oncogenic RAS or SRC or both. Oncogenic RAS or SRC activates KIAA0861 kinase activity. KIAA0861 is immunoprecipitated from cell extracts using a monoclonal antibody (e.g., 9E10) or a polyclonal antibody (e.g., from rabbit) specific for a unique peptide from KIAA0861. KIAA0861 is then resuspended in assay buffer containing GST-Mekl or GST-Mek2 and/or GST-ERK2. In addition, [γ³²P] ATP can be added to detect and/or quantify phosphorylation activity. Samples are incubated for 5-30 minutes at 30° C., and then the reaction is terminated by addition of EDTA. The samples are centrifuged and the supernatant fractions are collected. Phosphorylation activity is detected using one of two methods: (i) activity of GST-ERK2 kinase can be measured using MBP (myelin basic protein, a substrate for ERK) as substrate, or (ii) following incubation of immunoprecipitated KIAA0861 in reaction buffer containing GST-ERK and [γ³²P] ATP, transfer of labeled ATP to kinase-dead ERK can be quantified by a phosphor-imager or densitometer following PAGE separation of polypeptide products (phosphorylated and non-phosphorylated forms). These types of assays are described in Weber et al., Oncogene 19: 169-176 (2000); Mason et al., EMBO J. 18: 2137-2148 (1999); Marais et al., J. Biol. Chem. 272: 4378-4383 (1997); Marais et al., EMBO J. 14: 3136-3145 (1995).

Screening assays also are performed to identify molecules that regulate the interaction between a GEF, such as KIAA0861, and a GTPase. Such molecules can be identified using an assay for a GEF activity, such as guanine nucleotide exchange activity, binding to a guanine nucleotide-depleted site of a GTPase, or oncogenic transforming activity, or a TGPase activity such as GTP hydrolysis. In general, a compound having such an in vitro activity will be useful in vivo to modulate a biological pathway associated with a GTPase (e.g., to treat a pathological condition associated with the biological and cellular activities mentioned above). By way of illustration, the ways in which GEF regulators can be identified are described above and below in terms of Rho and KIAA0861. However, it is to be understood that such methods can be applied generally to other GEFs.

A guanine nucleotide exchange assay, e.g., as described in Hart et al., Nature, 354:311-314, 28 Nov. 1991, can be used to assay for the ability of a compound to regulate the interaction between Rho and KIAA0861. For example, Rho protein (recombinant, recombinant fusion protein, or isolated from natural sources) is labeled with tritiated-GDP. The tritiated-GDP-labeled Rho is then incubated with KIAA0861 and GTP under conditions in which nucleotide exchange occurs. The amount of tritiated-GDP that is retained by Rho is determined by separating bound GDP from free GDP, e.g., using a BA85 filter. The ability of a compound to regulate the interaction can be determined by adding the compound at a desired time to the incubation (e.g., before addition of KIAA0861, after addition of KIAA0861) and determining its effect on nucleotide exchange. Various agonist and antagonists of the interaction can be identified in this manner.

Binding to a guanine nucleotide-depleted site of Rho can be determined in various ways, e.g., as described in Hart et al., J. Biol. Chem. 269:62-65, 1994. Briefly, a Rho protein can be coupled to a solid support using various methods that one skilled in the art would know, e.g., using an antibody to Rho, a fusion protein between Rho and a marker protein, such as glutathione protein (GST), wherein the fusion is coupled to a solid support via the marker protein (such as glutathionine beads when GST is used), and the like. The Rho protein is converted to a guanine nucleotide depleted state (for effective conditions, see, e.g., Hart et al., J. Biol. Chem., 269:62-65, 1994) and incubated with, e.g., GDP, GTP γS, and GEF such as KIAA0861. The solid support is then separated and any protein on it run on a gel. A compound can be added at any time during the incubation (as described above) to determine its effect on the binding of GEF to Rho.

Modulation of oncogenic transforming activity by a KIAA0861, or derivatives thereof can be measured according to various known procedures, e.g., Eva and Aaronson, Nature, 316:273-275, 1985; Hart et al., J. Biol. Chem., 269:62-65, 1994. A compound can be added at any time during the method (e.g., pretreatment of cells; after addition of GEF, and the like) to determine its effect on the oncogenic transforming activity of KIAA0861. Various cell lines also can be used.

Other assays for Rho-mediated signal transduction can be accomplished according in analogy to procedures known in the art, e.g., as described in U.S. Pat. Nos. 5,141,851; 5,420,334; 5,436,128; and 5,482,954; WO94/16069; WO93/16179; WO91/15582; WO90/00607. In addition, peptides which inhibit the interaction, e.g., binding between KIAA0861 and a G-protein, such as RhoA, can be identified and prepared according to EP 496 162.

Included herein are methods of testing for and identifying an agent which modulates the guanine nucleotide exchange activity of a guanine nucleotide exchange factor, or a biologically-active fragment thereof, or which modulates the binding between a GEF, or a biologically-active fragment thereof, and a GTPase, or a biologically-active fragment thereof, to which it binds. The method comprises contacting the GEF and GTPase with an agent to be tested and then detecting the presence or amount of binding between the GEF and GTPase, or an activity of the GEF such as guanine nucleotide exchange activity. As discussed herein “modulating” refers to an agent that affects the activity or binding of a GEF such as KIAA0861. The binding or activity modulation can be affected in various ways, including inhibiting, blocking, preventing, increasing, enhancing, or promoting it. The binding or activity affected does not have to be achieved in a specific way, e.g., it can be competitive, noncompetitive, allosteric, sterically hindered, via cross-linking between the agent and the GEF or GTPase, or the like. The agent can act on either the GEF or GTPase. The agent can be an agonist, an antagonist, or a partial agonist or antagonist. The presence or amount of binding can be determined in various ways, e.g., directly or indirectly by assaying for an activity promoted or inhibited by the GEF, such as guanine nucleotide exchange, GTP hydrolysis, oncogenic transformation, and the like. Such assays are described above and below, and are also known in the art. The agent can be obtained and/or prepared from a variety of sources, including natural and synthetic. It can comprise, e.g., amino acids, lipids, carbohydrates, organic molecules, nucleic acids, inorganic molecules, or mixtures thereof. See, e.g., Hoeprich, Nature Biotechnology, 14:1311-1312, 1996, which describes an example of automated synthesis of organic molecules. The agent can be added simultaneously or sequentially. For example, the agent can be added to the GEF and then the resultant mixture can be further combined with the GTPase. The method can be carried out in liquid on isolated components, on a matrix (e.g., filter paper, nitrocellulose, agarose), in cells, on tissue sections, and the like. In accordance with the method, a GEF can bind to the GTPase, which binding will modulate some GTPase activity. For example, as discussed above and below, a KIAA0861 binds to Rho, causing guanine nucleotide dissociation. The effect can be directly on the binding site between the GEF and GTPase, or it can be allosteric, or it can be on only one component (e.g., on the GEF only) Assays for guanine nucleotide dissociation can be readily adapted to identify agents which regulate the activity of a GTPase. The method further relates to obtaining or producing agents which have been identified according to the above-described method. The present invention also relates to products identified in accordance with such methods. Various GEFs and GTPases can be employed, including KIAA0861, mSOS, SO, C3G, lsc, Dbl, Dbl-related proteins, polypeptides comprising one or more DH domains, CDC24, Tiam, Ost, Lbc, Vav, Ect2, Bcr, Abr, Rho (A, B, and C), Rac, Ras, CDC42, chimeras thereof, biologically-active fragments thereof, muteins thereof, and the like.

In general, an interaction can be determined by labeling the test molecule and/or the KIAA0861 molecule, where the label is covalently or non-covalently attached to the test molecule or KIAA0861 molecule. The label is sometimes a radioactive molecule such as ¹²⁵I, ¹³¹I, ³⁵S or ³H, which can be detected by direct counting of radioemission or by scintillation counting. Also, enzymatic labels such as horseradish peroxidase, alkaline phosphatase, or luciferase may be utilized where the enzymatic label can be detected by determining conversion of an appropriate substrate to product. Also, presence or absence of an interaction can be determined without labeling. For example, a microphysiometer (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indication of an interaction between a test molecule and KIAA0861 (McConnell, H. M. et al., Science 257: 1906-1912 (1992)).

In cell-based systems, cells typically include a KIAA0861 nucleic acid or polypeptide or variants thereof and are often of mammalian origin, although the cell can be of any origin. Whole cells, cell homogenates, and cell fractions (e.g., cell membrane fractions) can be subjected to analysis. Where interactions between a test molecule with a KIAA0861 polypeptide or variant thereof are monitored, soluble and/or membrane bound forms of the polypeptide or variant may be utilized. Where membrane-bound forms of the polypeptide are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate.

An interaction between two molecules also can be detected by monitoring fluorescence energy transfer (FET) (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos et al. U.S. Pat. No. 4,868,103). A fluorophore label on a first, “donor” molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, “acceptor” molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the “donor” polypeptide molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the “acceptor” molecule label may be differentiated from that of the “donor”. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the “acceptor” molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

In another embodiment, determining the presence or absence of an interaction between a test molecule and a KIAA0861 molecule can be effected by using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander & Urbaniczk, Anal. Chem. 63: 2338-2345 (1991) and Szabo et al., Curr. Opin. Struct. Biol. 5: 699-705 (1995)). “Surface plasmon resonance” or “BIA” detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

In another embodiment, the KIAA0861 molecule or test molecules are anchored to a solid phase. The KIAA0861 molecule/test molecule complexes anchored to the solid phase can be detected at the end of the reaction. The target KIAA0861 molecule is often anchored to a solid surface, and the test molecule, which is not anchored, can be labeled, either directly or indirectly, with detectable labels discussed herein.

It may be desirable to immobilize a KIAA0861 molecule, an anti-KIAA0861 antibody, or test molecules to facilitate separation of complexed from uncomplexed forms of KIAA0861 molecules and test molecules, as well as to accommodate automation of the assay. Binding of a test molecule to a KIAA0861 molecule can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion polypeptide can be provided which adds a domain that allows a KIAA0861 molecule to be bound to a matrix. For example, glutathione-S-transferase/KIAA0861 fusion polypeptides or glutathione-S-transferase/target fusion polypeptides can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivitized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target polypeptide or KIAA0861 polypeptide, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of KIAA0861 binding or activity determined using standard techniques.

Other techniques for immobilizing a KIAA0861 molecule on matrices include using biotin and streptavidin. For example, biotinylated KIAA0861 polypeptide or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactive with KIAA0861 polypeptide or test molecules but which do not interfere with binding of the KIAA0861 polypeptide to its test molecule. Such antibodies can be derivitized to the wells of the plate, and unbound target or KIAA0861 polypeptide trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the KIAA0861 polypeptide or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the KIAA0861 polypeptide or test molecule.

Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G., and Minton, A. P., Trends Biochem Sci August; 18(8): 284-7 (1993)); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel et al, eds. Current Protocols in Molecular Biology, J. Wiley: New York (1999)); and immunoprecipitation (see, for example, Ausubel, F. et al., eds. Current Protocols in Molecular Biology, J. Wiley: New York (1999)). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, J. Mol. Recognit. Winter; 11(1-6): 141-8 (1998); Hage & Tweed, J. Chromatogr. B Biomed. Sci. Appl. October 10; 699 (1-2): 499-525 (1997)). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

In another embodiment, modulators of KIAA0861 expression are identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of KIAA0861 mRNA or polypeptide evaluated relative to the level of expression of KIAA0861 mRNA or polypeptide in the absence of the candidate compound. When expression of KIAA0861 mRNA or polypeptide is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of KIAA0861 mRNA or polypeptide expression. Alternatively, when expression of KIAA0861 mRNA or polypeptide is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of KIAA0861 mRNA or polypeptide expression. The level of KIAA0861 mRNA or polypeptide expression can be determined by methods described herein for detecting KIAA0861 mRNA or polypeptide.

In another embodiment, binding partners that interact with a KIAA0861 molecule are detected. The KIAA0861 molecules can interact with one or more cellular or extracellular macromolecules, such as polypeptides, in vivo, and these molecules that interact with KIAA0861 molecules are referred to herein as “binding partners.” Molecules that disrupt such interactions can be useful in regulating the activity of the target gene product. Such molecules can include, but are not limited to molecules such as antibodies, peptides, and small molecules. Target genes/products for use in this embodiment often are the KIAA0861 genes herein identified. In an alternative embodiment, provided is a method for determining the ability of the test compound to modulate the activity of a KIAA0861 polypeptide through modulation of the activity of a downstream effector of a KIAA0861 target molecule. For example, the activity of the effector molecule on an appropriate target can be determined, or the binding of the effector to an appropriate target can be determined, as previously described.

To identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner(s), e.g., a substrate, a reaction mixture containing the target gene product and the binding partner is prepared, under conditions and for a time sufficient, to allow the two products to form complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the target gene and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target gene product and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases where it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene products.

These assays can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below.

In a heterogeneous assay system, either the target gene product or the interactive cellular or extracellular binding partner, is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly. The anchored species can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.

In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that disrupt preformed complexes can be identified.

In an alternate embodiment, a homogeneous assay can be used. For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared in that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified.

Also, binding partners of KIAA0861 molecules can be identified in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., Cell 72:223-232 (1993); Madura et al, J. Biol. Chem. 268: 12046-12054 (1993); Bartel et al., Biotechniques 14: 920-924 (1993); Iwabuchi et al., Oncogene 8: 1693-1696 (1993); and Brent WO94/10300), to identify other polypeptides, which bind to or interact with KIAA0861 (“KIAA0861-binding polypeptides” or “KIAA0861-bp”) and are involved in KIAA0861 activity. Such KIAA0861-bps can be activators or inhibitors of signals by the KIAA0861 polypeptides or KIAA0861 targets as, for example, downstream elements of a KIAA0861-mediated signaling pathway.

A two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a KIAA0861 polypeptide is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified polypeptide (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. (Alternatively the: KIAA0861 polypeptide can be the fused to the activator domain.) If the “bait” and the “prey” polypeptides are able to interact, in vivo, forming a KIAA0861-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the polypeptide which interacts with the KIAA0861 polypeptide.

Candidate therapeutics for treating breast cancer are identified from a group of test molecules that interact with a KIAA0861 nucleic acid or polypeptide. Test molecules are normally ranked according to the degree with which they interact or modulate (e.g., agonize or antagonize) DNA replication and/or processing, RNA transcription and/or processing, polypeptide production and/or processing, and/or function of KIAA0861 molecules, for example, and then top ranking modulators are selected. In a preferred embodiment, the candidate therapeutic (i.e., test molecule) acts as a KIAA0861 antagonist. Also, pharmacogenomic information described herein can determine the rank of a modulator. Candidate therapeutics typically are formulated for administration to a subject.

Therapeutic Treatments

Formulations or pharmaceutical compositions typically include in combination with a pharmaceutically acceptable carrier, a compound, an antisense nucleic acid, a ribozyme, an antibody, a binding partner that interacts with a KIAA0861 polypeptide, a KIAA0861 nucleic acid, or a fragment thereof. The formulated molecule may be one that is identified by a screening method described above. Also, formulations may comprise a KIAA0861 polypeptide or fragment thereof, where the KIAA0861 polypeptide is able to bind to a Rho GTPase but unable to catalyze GDP-GTP exchange reactions of Rho proteins. As used herein, the term “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride sometimes are included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation often utilized are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. Molecules can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, active molecules are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Molecules which exhibit high therapeutic indices often are utilized. While molecules that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such molecules often lies within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any molecules used in the methods described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, sometimes about 0.01 to 25 mg/kg body weight, often about 0.1 to 20 mg/kg body weight, and more often about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, sometimes between 2 to 8 weeks, often between about 3 to 7 weeks, and more often for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment, or sometimes can include a series of treatments.

With regard to polypeptide formulations, featured herein is a method for treating breast cancer in a subject, which comprises contacting one or more cells in the subject with a polypeptide that interacts with a KIAA0861 polypeptide and inhibits its guanine nucleotide exchange factor activity.

For antibodies, a dosage of 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg) is often utilized. If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is often appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al., J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193 (1997).

Antibody conjugates can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a polypeptide such as tumor necrosis factor, .alpha.-interferon, .beta.-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors. Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.

For compounds, exemplary doses include milligram or microgram amounts of the compound per kilogram of subject or sample weight, for example, about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid described herein, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

KIAA0861 nucleic acid molecules can be inserted into vectors and used in gene therapy methods for treating breast cancer. Featured herein is a method for treating breast cancer in a subject, which comprises contacting one or more cells in the subject with a first KIAA0861 nucleic acid, where genomic DNA in the subject comprises a second KIAA0861 nucleic acid comprising one or more polymorphic variations associated with breast cancer, and where the first KIAA0861 nucleic acid comprises fewer polymorphic variations associated with breast cancer. The first and second KIAA0861 nucleic acids typically comprise a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1-3; a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence of SEQ ID NO: 4 or 5; a nucleotide sequence that is 90% or more identical to the nucleotide sequence of SEQ ID NO: 1-3, and a nucleotide sequence which encodes a polypeptide that is 90% identical to an amino acid sequence of SEQ ID NO: 4 or 5. The second KIAA0861 nucleic acid also may be a fragment of the foregoing comprising one or more polymorphic variations. The subject often is a human.

Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al., (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). Pharmaceutical preparations of gene therapy vectors can include a gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells (e.g., retroviral vectors) the pharmaceutical preparation can include one or more cells which produce the gene delivery system. Examples of gene delivery vectors are described herein.

Pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

Pharmaceutical compositions of active ingredients can be administered by any of the paths described herein for therapeutic and prophylactic methods for treating breast cancer. With regard to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from pharmacogenomic analyses described herein. As used herein, the term “treatment” is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.

Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the KIAA0861 aberrance, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of KIAA0861 aberrance, for example, a KIAA0861 molecule, KIAA0861 agonist, or KIAA0861 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

As discussed, successful treatment of KIAA0861 disorders can be brought about by techniques that serve to inhibit the expression or activity of target gene products. For example, compounds (e.g., an agent identified using an assays described above) that exhibit negative modulatory activity can be used to prevent and/or treat breast cancer. Such molecules can include, but are not limited to peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab′)2 and FAb expression library fragments, scFV molecules, and epitope-binding fragments thereof).

Further, antisense and ribozyme molecules that inhibit expression of the target gene can also be used to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. Still further, triple helix molecules can be utilized in reducing the level of target gene activity. Antisense, ribozyme and triple helix molecules are discussed above.

It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method. Alternatively, in instances where the target gene encodes an extracellular polypeptide, normal target gene polypeptide often is co-administered into the cell or tissue to maintain the requisite level of cellular or tissue target gene activity.

Another method by which nucleic acid molecules may be utilized in treating or preventing a disease characterized by KIAA0861 expression is through the use of aptamer molecules specific for KIAA0861 polypeptide. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to polypeptide ligands (see, e.g., Osborne, et al, Curr. Opin. Chem. Biol. 1(1): 5-9 (1997); and Patel, D. J., Curr. Opin. Chem. Biol. June; 1(1): 32-46 (1997)). Since nucleic acid molecules may in many cases be more conveniently introduced into target cells than therapeutic polypeptide molecules may be, aptamers offer a method by which KIAA0861 polypeptide activity may be specifically decreased without the introduction of drugs or other molecules which may have pluripotent effects.

Antibodies can be generated that are both specific for target gene product and that reduce target gene product activity. Such antibodies may, therefore, by administered in instances whereby negative modulatory techniques are appropriate for the treatment of KIAA0861 disorders. For a description of antibodies, see the Antibody section above.

In circumstances where injection of an animal or a human subject with a KIAA0861 polypeptide or epitope for stimulating antibody production is harmful to the subject, it is possible to generate an immune response against KIAA0861 through the use of anti-idiotypic antibodies (see, for example, Herlyn, D., Ann. Med.; 31(1): 66-78 (1999); and Bhattacharya-Chatterjee & Foon, Cancer Treat. Res.; 94: 51-68 (1998)). If an anti-idiotypic antibody is introduced into a mammal or human subject, it should stimulate the production of anti-anti-idiotypic antibodies, which should be specific to the KIAA0861 polypeptide. Vaccines directed to a disease characterized by KIAA0861 expression may also be generated in this fashion.

In instances where the target antigen is intracellular and whole antibodies are used, internalizing antibodies may be utilized. Lipofectin or liposomes can be used to deliver the antibody or a fragment of the Fab region that binds to the target antigen into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target antigen often is utilized. For example, peptides having an amino acid sequence corresponding to the Fv region of the antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intracellular target antigens can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see e.g., Marasco et al., Proc. Natl. Acad. Sci. USA 90: 7889-7893 (1993)).

KIAA0861 molecules and compounds that inhibit target gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to prevent, treat or ameliorate KIAA0861 disorders. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of the disorders.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit large therapeutic indices often are utilized. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds often lies within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in a method described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

Another example of effective dose determination for an individual is the ability to directly assay levels of “free” and “bound” compound in the serum of the test subject. Such assays may utilize antibody mimics and/or “biosensors” that have been created through molecular imprinting techniques. The compound which is able to modulate KIAA0861 activity is used as a template, or “imprinting molecule”, to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix which contains a repeated “negative image” of the compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell et al., Current Opinion in Biotechnology 7: 89-94 (1996) and in Shea, Trends in Polymer Science 2: 166-173 (1994). Such “imprinted” affinity matrixes are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example of the use of such matrixes in this way can be seen in Vlatakis, et al, Nature 361: 645-647 (1993). Through the use of isotope-labeling, the “free” concentration of compound which modulates the expression or activity of KIAA0861 can be readily monitored and used in calculations of IC₅₀. Such “imprinted” affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes can be readily assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC₅₀. A rudimentary example of such a “biosensor” is discussed in Kriz et al., Analytical Chemistry 67: 2142-2144 (1995).

Provided herein are methods of modulating KIAA0861 expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method involves contacting a cell with a KIAA0861 or agent that modulates one or more of the activities of KIAA0861 polypeptide activity associated with the cell. An agent that modulates KIAA0861 polypeptide activity can be an agent as described herein, such as a nucleic acid or a polypeptide, a naturally-occurring target molecule of a KIAA0861 polypeptide (e.g., a KIAA0861 substrate or receptor), a KIAA0861 antibody, a KIAA0861 agonist or antagonist, a peptidomimetic of a KIAA0861 agonist or antagonist, or other small molecule.

In one embodiment, the agent stimulates one or more KIAA0861 activities. Examples of such stimulatory agents include active KIAA0861 polypeptide and a nucleic acid molecule encoding KIAA0861. In another embodiment, the agent inhibits one or more KIAA0861 activities. Examples of such inhibitory agents include antisense KIAA0861 nucleic acid molecules, anti-KIAA0861 antibodies, and KIAA0861 inhibitors, and competitive inhibitors that target Rho family GTP-binding proteins that are regulated by KIAA0861. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, provided are methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a KIAA0861 polypeptide or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) KIAA0861 expression or activity. In a preferred embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that inhibits KIAA0861 expression or activity (e.g., a KIAA0861 activity may include catalyzing the exchange of Rho-bound GDP for GTP). In another embodiment, the method involves administering a KIAA0861 polypeptide or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted KIAA0861 expression or activity.

Stimulation of KIAA0861 activity is desirable in situations in which KIAA0861 is abnormally downregulated and/or in which increased KIAA0861 activity is likely to have a beneficial effect. For example, stimulation of KIAA0861 activity is desirable in situations in which a KIAA0861 is downregulated and/or in which increased KIAA0861 activity is likely to have a beneficial effect. Likewise, inhibition of KIAA0861 activity is desirable in situations in which KIAA0861 is abnormally upregulated and/or in which decreased KIAA0861 activity is likely to have a beneficial effect.

Methods of Treatment

In another aspect, provided are methods for identifying a predisposition to cancer in an individual as described herein and, if a genetic predisposition is identified, treating that individual to delay or reduce or prevent the development of cancer. Such a procedure can be used to treat breast cancer. Optionally, treating an individual for cancer may include inhibiting cellular proliferation, inhibiting metastasis, inhibiting invasion, or preventing tumor formation or growth as defined herein. Suitable treatments to prevent or reduce or delay breast cancer focus on inhibiting additional cellular proliferation, inhibiting metastasis, inhibiting invasion, and preventing further tumor formation or growth. Treatment usually includes surgery followed by radiation therapy. Surgery may be a lumpectomy or a mastectomy (e.g., total, simple or radical). Even if the doctor removes all of the cancer that can be seen at the time of surgery, the patient may be given radiation therapy, chemotherapy, or hormone therapy after surgery to try to kill any cancer cells that may be left. Radiation therapy is the use of x-rays or other types of radiation to kill cancer cells and shrink tumors. Radiation therapy may use external radiation (using a machine outside the body) or internal radiation. Chemotherapy is the use of drugs to kill cancer cells. Chemotherapy may be taken by mouth, or it may be put into the body by inserting a needle into a vein or muscle. Hormone therapy often focuses on estrogen and progesterone, which are hormones that affect the way some cancers grow. If tests show that the cancer cells have estrogen and progesterone receptors (molecules found in some cancer cells to which estrogen and progesterone will attach), hormone therapy is used to block the way these hormones help the cancer grow. Hormone therapy with tamoxifen is often given to patients with early stages of breast cancer and those with metastatic breast cancer. Other types of treatment being tested in clinical trials include sentinel lymph node biopsy followed by surgery and high-dose chemotherapy with bone marrow transplantation and peripheral blood stem cell transplantation. Any preventative/therapeutic treatment known in the art may be prescribed and/or administered, including, for example, surgery, chemotherapy and/or radiation treatment, and any of the treatments may be used in combination with one another to treat or prevent breast cancer (e.g., surgery followed by radiation therapy).

Rho-proteins function as binary switches cycling between a biologically inactive GDP-bound state and a biologically active GTP-bound state. Conversion to the GTP-bound form is mediated by the actions of Rho-GEFs, which stimulate the dissociation of bound GDP, thus providing an opportunity for GTP to bind. Since most Rho proteins exhibit biological activity only when in the GTP-bound state, RhoGEFs, such as KIAA0861, are thought to be Rho activators. Regulation of RhoGEFs are believed to be the result of several regulatory modes involving intra or inter-molecular interactions (Zheng, Y. Trends Biochem. Sci. 26(12): 724-732 (2001)). A first possible RhoGEF regulatory mode is through the intramolecular interaction between DH and PH domains. A second possible RhoGEF regulatory mode is through the intramolecular interaction of a regulatory domain with the DH or PH domain such that the regulatory domain imposes a constraint on the normal DH and/or PH domain functions. A third possible mode involves oligomerization through an intermolecular interaction between Dh domains, thus allowing for the recruitment of multiple Rho substrates into one signaling complex. A final possible mode involves the recruitment of inhibitory cellular factors that suppress GEF activity and help maintain the basal, inactive state. Regulatory molecules known to affect RhoGEF activity include kinases, lipid products from kinase reactions such as phosphoinositol phosphates, and cytoskelelton proteins such as ankyrin (Zheng, Y. Trends Biochem. Sci. 26(12): 724-732 (2001)). Deregulation of any of these regulatory mechanisms due to a specific mutation in KIAA0861 can lead to a Rho protein that is always biologically active, thus leading to oncogenesis.

Thus, featured herein are methods of regulating a biological pathway in which a GTPase is involved, particularly pathological conditions, e.g., cell proliferation (e.g., cancer), growth control, morphogenesis, stress fiber formation, and integrin-mediated interactions, such as embryonic development, tumor cell growth and metastasis, programmed cell death, hemostatis, leukocyte homing and activation, bone resorption, clot retraction, and the response of cells to mechanical stress. See, e.g., Clarke and Brugge, Science 268:233-239, 1995; Bussey, Science, 272:225-226, 1996. Thus, all aspects of modulating an activity of a Rho polypeptide is included herein, which often comprises administering an effective amount of a compound which modulates the activity of a Rho polypeptide, or an effective amount of a nucleic acid which codes for a KIAA0861 polypeptide or a biologically-active fragment thereof. The activity of Rho which is modulated can include: GTP binding, GDP binding, GTPase activity, integrin binding, coupling or binding or Rho to receptor or effector-like molecules (such as integrins, growth factor receptors, tyrosine kinases, PI-3K, PIP-5K, and the like). See, e.g., Clarke and Brugge, Science 268:233-239, 1995. The activity can be modulated by increasing, reducing, antagonizing, promoting, and the like, of Rho. The modulation of Rho can be measured by assayed routinely for GTP hydrolysis, PI(4,5)biphosphate, binding to KIAA0861 or a similar assay such as the one described in Example 11. An effective amount is any amount which, when administered, modulates the Rho activity. The activity can be modulated in a cell, a tissue, a whole organism, in situ, in vitro (test tube, a solid support, and the like), in vivo, or in any desired environment.

Also provided are methods of preventing or treating breast cancer comprising providing an individual in need of such treatment with a GEF inhibitor that reduces or blocks the dysregulated guanine nucleotide exchange function of the GEF in the subject. In some embodiments, it is preferable to specifically reduce or block the dysregulated guanine nucleotide exchange function of KIAA0861 by administering a KIAA0861 inhibitor to the subject in need thereof (e.g., an inhibitor that inhibits the activity of KIAA0861 more than the activity of another GEF). GEF inhibitors and KIAA0861 specific inhibitors sometimes bind to a GEF or KIAA0861 polypeptide or interact with another peptide and reduce the guanine nucleotide exchange function of the GEF or KIAA0861. Also included are methods of reducing or blocking the guanine nucleotide exchange function of KIAA0861 by introducing point mutations into the catalytic domain of KIAA0861 to inhibit its GDP-GTP exchange activity. In the embodiments described above, treating or preventing breast cancer are specifically directed to reducing or inhibiting breast cancer cell metastasis. Thus, featured are methods for reducing or inhibiting breast cancer cell metastasis by inhibiting a GEF or specifically inhibiting KIAA0861. Data shown herein demonstrates that inhibition of KIAA0861 can inhibit cancer metastasis (e.g., see siRNA results herein).

The examples set forth below are intended to illustrate but not limit the invention.

EXAMPLES

In the following studies a group of subjects were selected according to specific parameters relating to breast cancer. Nucleic acid samples obtained from individuals in the study group were subjected to genetic analysis, which identified associations between breast cancer and a polymorphism in the KIAA0861 gene on chromosome three. Methods are described for producing KIAA0861 polypeptide and KIAA0861 polypeptide variants in vitro or in vivo, KIAA0861 nucleic acids or polypeptides and variants thereof are utilized for screening test molecules for those that interact with KIAA0861 molecules. Test molecules identified as interactors with KIAA0861 molecules and KIAA0861 variants are further screened in vivo to determine whether they treat breast cancer.

Example 1 Samples and Pooling Strategies Sample Selection

Blood samples were collected from individuals diagnosed with breast cancer, which were referred to as case samples. Also, blood samples were collected from individuals not diagnosed with breast cancer as gender and age-matched controls. All of the samples were of German/German descent. A database was created that listed all phenotypic trait information gathered from individuals for each case and control sample. Genomic DNA was extracted from each of the blood samples for genetic analyses.

DNA Extraction from Blood Samples

Six to ten milliliters of whole blood was transferred to a 50 ml tube containing 27 ml of red cell lysis solution (RCL). The tube was inverted until the contents were mixed. Each tube was incubated for 10 minutes at room temperature and inverted once during the incubation. The tubes were then centrifuged for 20 minutes at 3000×g and the supernatant was carefully poured off. 100-200 μl of residual liquid was left in the tube and was pipetted repeatedly to resuspend the pellet in the residual supernatant. White cell lysis solution (WCL) was added to the tube and pipetted repeatedly until completely mixed. While no incubation was normally required, the solution was incubated at 37° C. or room temperature if cell clumps were visible after mixing until the solution was homogeneous. 2 ml of protein precipitation was added to the cell lysate. The mixtures were vortexed vigorously at high speed for 20 sec to mix the protein precipitation solution uniformly with the cell lysate, and then centrifuged for 10 minutes at 3000×g. The supernatant containing the DNA was then poured into a clean 15 ml tube, which contained 7 ml of 100% isopropanol. The samples were mixed by inverting the tubes gently until white threads of DNA were visible. Samples were centrifuged for 3 minutes at 2000×g and the DNA was visible as a small white pellet. The supernatant was decanted and 5 ml of 70% ethanol was added to each tube. Each tube was inverted several times to wash the DNA pellet, and then centrifuged for 1 minute at 2000×g. The ethanol was decanted and each tube was drained on clean absorbent paper. The DNA was dried in the tube by inversion for 10 minutes, and then 1000 μl of 1×TE was added. The size of each sample was estimated, and less TE buffer was added during the following DNA hydration step if the sample was smaller. The DNA was allowed to rehydrate overnight at room temperature, and DNA samples were stored at 2-8° C.

DNA was quantified by placing samples on a hematology mixer for at least 1 hour. DNA was serially diluted (typically 1:80, 1:160, 1:320, and 1:640 dilutions) so that it would be within the measurable range of standards. 125 μl of diluted DNA was transferred to a clear U-bottom microtiter plate, and 125 μl of 1×TE buffer was transferred into each well using a multichannel pipette. The DNA and 1×TE were mixed by repeated pipetting at least 15 times, and then the plates were sealed. 50 μl of diluted DNA was added to wells A5-H12 of a black flat bottom microtiter plate. Standards were inverted six times to mix them, and then 50 μl of 1×TE buffer was pipetted into well A1, 1000 ng/ml of standard was pipetted into well A2, 500 ng/ml of standard was pipetted into well A3, and 250 ng/ml of standard was pipetted into well A4. PicoGreen (Molecular Probes, Eugene, Oreg.) was thawed and freshly diluted 1:200 according to the number of plates that were being measured. PicoGreen was vortexed and then 50 μl was pipetted into all wells of the black plate with the diluted DNA. DNA and PicoGreen were mixed by pipetting repeatedly at least 10 times with the multichannel pipette. The plate was placed into a Fluoroskan Ascent Machine (microplate fluorometer produced by Labsystems) and the samples were allowed to incubate for 3 minutes before the machine was run using filter pairs 485 nm excitation and 538 nm emission wavelengths. Samples having measured DNA concentrations of greater than 450 ng/μl were re-measured for conformation. Samples having measured DNA concentrations of 20 ng/μl or less were re-measured for confirmation.

Pooling Strategies

Samples were placed into one of two groups based on disease status. The two groups were female case groups and female control groups. A select set of samples from each group were utilized to generate pools, and one pool was created for each group. Each individual sample in a pool was represented by an equal amount of genomic DNA. For example, where 25 ng of genomic DNA was utilized in each PCR reaction and there were 200 individuals in each pool, each individual would provide 125 pg of genomic DNA. Inclusion or exclusion of samples for a pool was based upon the following criteria: the sample was derived from an individual characterized as Caucasian; the sample was derived from an individual of German paternal and maternal descent; the database included relevant phenotype information for the individual; case samples were derived from individuals diagnosed with breast cancer; control samples were derived from individuals free of cancer and no family history of breast cancer; and sufficient genomic DNA was extracted from each blood sample for all allelotyping and genotyping reactions performed during the study. Phenotype information included pre- or post-menopausal, familial predisposition, country or origin of mother and father, diagnosis with breast cancer (date of primary diagnosis, age of individual as of primary diagnosis, grade or stage of development, occurrence of metastases, e.g., lymph node metastases, organ metastases), condition of body tissue (skin tissue, breast tissue, ovary tissue, peritoneum tissue and myometrium), method of treatment (surgery, chemotherapy, hormone therapy, radiation therapy). Samples that met these criteria were added to appropriate pools based on gender and disease status.

The selection process yielded the pools set forth in Table 1, which were used in the studies that follow:

TABLE 1 Female CASE Female CONTROL Pool size 272 276 (Number) Pool Criteria case control (ex: case/control) Mean Age 59.6 55.4 (ex: years)

Example 2 Association of Polymorphic Variants with Breast Cancer

A whole-genome screen was performed to identify particular SNPs associated with occurrence of breast cancer. As described in Example 1, two sets of samples were utilized, which included samples from female individuals having breast cancer (breast cancer cases) and samples from female individuals not having cancer (female controls). The initial screen of each pool was performed in an allelotyping study, in which certain samples in each group were pooled. By pooling DNA from each group, an allele frequency for each SNP in each group was calculated. These allele frequencies were then compared to one another. Particular SNPs were considered as being associated with breast cancer when allele frequency differences calculated between case and control pools were statistically significant. SNP disease association results obtained from the allelotyping study were then validated by genotyping each associated SNP across all samples from each pool. The results of the genotyping were then analyzed, allele frequencies for each group were calculated from the individual genotyping results, and a p value was calculated to determine whether the case and control groups had statistically significantly differences in allele frequencies for a particular SNP. When the genotyping results agreed with the original allelotyping results, the SNP disease association was considered validated at the genetic level.

It was discovered that females having a cytosine at position 33106 of SEQ ID NO: 1 were predisposed to breast cancer.

SNP Panel Used for Genetic Analyses

A whole-genome SNP screen began with an initial screen of approximately 25,000 SNPs over each set of disease and control samples using a pooling approach. The pools studied in the screen are described in Example 1. The SNPs analyzed in this study were part of a set of 25,488 SNPs confirmed as being statistically polymorphic as each is characterized as having a minor allele frequency of greater than 10%. The SNPs in the set reside in genes or in close proximity to genes, and many reside in gene exons. Specifically, SNPs in the set are located in exons, introns, and within 5,000 base-pairs upstream of a transcription start site of a gene. In addition, SNPs were selected according to the following criteria: they are located in ESTs; they are located in Locuslink or Ensemble genes; and they are located in Genomatix promoter predictions. SNPs in the set also were selected on the basis of even spacing across the genome, as depicted in Table 2.

A case-control study design using a whole genome association strategy involving approximately 28,000 single nucleotide polymorphisms (SNPs) was employed. Approximately 25,000 SNPs were evenly spaced in gene-based regions of the human genome with a median inter-marker distance of about 40,000 base pairs. Additionally, approximately 3,000 SNPs causing amino acid substitutions in genes described in the literature as candidates for various diseases were used. The case-control study samples were of female German origin (German paternal and maternal descent) 548 individuals were equally distributed in two groups (female controls and female cases). The whole genome association approach was first conducted on 2 DNA pools representing the 2 groups. Significant markers were confirmed by individual genotyping.

TABLE 2 General Statistics Spacing Statistics Total # of SNPs 28,532 Median   34,424 bp # of Exonic SNPs  8,497 (30%) Minimum*    1,000 bp # SNPs with refSNP ID 26,625 (93%) Maximum* 3,000,000 bp Gene Coverage 23,874 Mean   122,412 bp Chromosome Coverage All Std Deviation    354, bp *Excludes outliers

Allelotyping and Genotyping Results

The genetic studies summarized above and described in more detail below identified an allelic variant associated with breast cancer, set forth in Table 3.

TABLE 3 Position SNP Chromosome in SEQ ID Contig Contig Sequence Sequence Allelic Reference Chromosome Position NO: 1 Identification Position Identification Locus Position Variability 2001449 3 184049849 48563 NT_005612 89424094 NM_015078 KIAA0861 intron G/C

Assay for Verifying, Allelotyping, and Genotyping SNPs

A MassARRAY™ system (Sequenom, Inc.) was utilized to perform SNP genotyping in a high-throughput fashion. This genotyping platform was complemented by a homogeneous, single-tube assay method (hME™ or homogeneous MassEXTEND™ (Sequenom, Inc.)) in which two genotyping primers anneal to and amplify a genomic target surrounding a polymorphic site of interest. A third primer (the MassEXTEND™ primer), which is complementary to the amplified target up to but not including the polymorphism, was then enzymatically extended one or a few bases through the polymorphic site and then terminated.

For each polymorphism, SpectroDESIGNER™ software (Sequenom, Inc.) was used to generate a set of PCR primers and a MassEXTEND™ primer was used to genotype the polymorphism. Table 4 shows PCR primers and Table 5 shows extension primers used for analyzing polymorphisms. The initial PCR amplification reaction was performed in a 5 μl total volume containing 1×PCR buffer with 1.5 mM MgCl₂ (Qiagen), 200 μM each of dATP, dGTP, dCTP, dTTP (Gibco-BRL), 2.5 ng of genomic DNA, 0.1 units of HotStar DNA polymerase (Qiagen), and 200 nM each of forward and reverse PCR primers specific for the polymorphic region of interest.

TABLE 4 PCR Primers Reference Forward PCR primer Reverse PCR primer SNP ID (SEQ ID NO: 7) (SEQ ID NO: 8) rs2001449 ATGTCAAGTGCACCCACA AGGAAGAAACTGACGGAAGG TG

Samples were incubated at 95° C. for 15 minutes, followed by 45 cycles of 95° C. for 20 seconds, 56° C. for 30 seconds, and 72° C. for 1 minute, finishing with a 3 minute final extension at 72° C. Following amplification, shrimp alkaline phosphatase (SAP) (0.3 units in a 2 μl volume) (Amersham Pharmacia) was added to each reaction (total reaction volume was 7 μl) to remove any residual dNTPs that were not consumed in the PCR step. Samples were incubated for 20 minutes at 37° C., followed by 5 minutes at 85° C. to denature the SAP.

Once the SAP reaction was complete, a primer extension reaction was initiated by adding a polymorphism-specific MassEXTEND™ primer cocktail to each sample. Each MassEXTEND™ cocktail included a specific combination of dideoxynucleotides (ddNTPs) and deoxynucleotides (dNTPs) used to distinguish polymorphic alleles from one another. In Table 5, ddNTPs are shown and the fourth nucleotide not shown is the dNTP.

TABLE 5 Reference Extend Probe Term SNP ID (SEQ ID NO: 9) Mix rs2001449 CACATGCCTGCTCGCCCCC ACT

The MassEXTEND™ reaction was performed in a total volume of 9 μl, with the addition of 1× ThermoSequenase buffer, 0.576 units of ThermoSequenase (Amersham Pharmacia), 600 nM MassEXTEND™ primer, 2 mM of ddATP and/or ddCTP and/or ddGTP and/or ddTTP, and 2 mM of dATP or dCTP or dGTP or dTTP. The deoxy nucleotide (dNTP) used in the assay normally was complementary to the nucleotide at the polymorphic site in the amplicon. Samples were incubated at 94° C. for 2 minutes, followed by 55 cycles of 5 seconds at 94° C., 5 seconds at 52° C., and 5 seconds at 72° C.

Following incubation, samples were desalted by adding 16 μl of water (total reaction volume was 25 μl), 3 mg of SpectroCLEAN™ sample cleaning beads (Sequenom, Inc.) and allowed to incubate for 3 minutes with rotation. Samples were then robotically dispensed using a piezoelectric dispensing device (SpectroJET™ (Sequenom, Inc.)) onto either 96-spot or 384-spot silicon chips containing a matrix that crystallized each sample (SpectroCHIP® (Sequenom, Inc.)). Subsequently, MALDI-TOF mass spectrometry (Biflex and Autoflex MALDI-TOF mass spectrometers (Bruker Daltonics) can be used) and SpectroTYPER RT™ software (Sequenom, Inc.) were used to analyze and interpret the SNP genotype for each sample.

Genetic Analysis

Variations identified in the KIAA0861 gene are represented by SEQ ID NO: 1 at position 33106. Minor allelic frequencies for these polymorphisms was verified as being 10% or greater by determining the allelic frequencies using the extension assay described above in a group of samples isolated from 92 individuals originating from the state of Utah in the United States, Venezuela and France (Coriell cell repositories).

Genotyping results are shown for female pools in Table 6A and 6B. Table 6A shows the original genotyping results and Table 6B shows the genotyped results re-analyzed to remove duplicate individuals from the cases and controls (i.e., individuals who were erroneously included more than once as either cases or controls). Therefore, Table 6B represents a more accurate measure of the allele frequencies for this particular SNP. In the subsequent tables, “AF” refers to allelic frequency; and “F case” and “F control” refer to female case and female control groups, respectively.

TABLE 6A Reference SNP ID AF F case AF F control p-value Odds Ratio rs2001449 G = 0.703 G = 0.780 0.0040 1.49 C = 0.297 C = 0.220

TABLE 6B Reference Odds SNP ID AF F case AF F control p-value Ratio rs2001449 G = 0.693 G = 0.782 0.0012 1.59 C = 0.307 C = 0.218

As can be seen in Tables 6A and 6B, a cytosine at position 33106 were more common in the female breast cancer group. Genotyping results were considered significant with a calculated p-value of less than 0.05 for genotype results.

Odds ratio results are shown in Tables 6A and 6B. An odds ratio is an unbiased estimate of relative risk which can be obtained from most case-control studies. Relative risk (RR) is an estimate of the likelihood of disease in the exposed group (susceptibility allele or genotype carriers) compared to the unexposed group (not carriers). It can be calculated by the following equation:

RR=IA/Ia

IA is the incidence of disease in the A carriers and Ia is the incidence of disease in the non-carriers.

RR>1 indicates the A allele increases disease susceptibility.

RR<1 indicates the a allele increases disease susceptibility.

For example, RR=1.5 indicates that carriers of the A allele have 1.5 times the risk of disease than non-carriers, i.e., 50% more likely to get the disease.

Case-control studies do not allow the direct estimation of IA and Ia, therefore relative risk cannot be directly estimated. However, the odds ratio (OR) can be calculated using the following equation:

OR=(nDAnda)/(ndAnDa)=pDA(1−pdA)/pdA(1−pDA), or

OR=((case f)/(1−case f))/((control f)/(1−control f)), where f=susceptibility allele frequency.

An odds ratio can be interpreted in the same way a relative risk is interpreted and can be directly estimated using the data from case-control studies, i.e., case and control allele frequencies. The higher the odds ratio value, the larger the effect that particular allele has on the development of breast cancer. Possessing an allele associated with a relatively high odds ratio translates to having a higher risk of developing or having breast cancer.

Example 3 Samples and Pooling Strategies for the Replication Samples

SNP reference number rs2001449 was genotyped again in a collection of replication samples to further validate its association with breast cancer. Like the original study population described in Examples 1 and 2, the replication samples consisted of females diagnosed with breast cancer (cases) and females without cancer (controls). The case and control samples were selected and genotyped as described below.

Samples were placed into one of two groups based on disease status. The two groups were female case groups and female control groups. A select set of samples from each group were utilized to generate pools, and one pool was created for each group. Each individual sample in a pool was represented by an equal amount of genomic DNA. For example, where 25 ng of genomic DNA was utilized in each PCR reaction and there were 200 individuals in each pool, each individual would provide 125 pg of genomic DNA. Inclusion or exclusion of samples for a pool was based upon the following criteria: the sample was derived from a female individual characterized as Caucasian; case samples were derived from individuals diagnosed with breast cancer; control samples were derived from individuals free of cancer and no family history of breast cancer; and sufficient genomic DNA was extracted from each blood sample for all allelotyping and genotyping reactions performed during the study. Samples that met these criteria were added to appropriate pools based on gender and disease status.

The selection process yielded the “Griffith” samples set forth in Table 7A and the “Kiechle” samples set forth in Table 7B, which were used in the studies that follow:

TABLE 7A Female CASE Female CONTROL Pool size 190 190 (Number) Pool Criteria case control (ex: case/control) Mean Age 64.5 ** (ex: years) ** Each case was matched by a control within 5 years of age of the case.

TABLE 7B Female CASE Female CONTROL Pool size 195 153 (Number) Pool Criteria case control (ex: case/control)

The replication genotyping results are shown in Table 8A for the Griffith samples and in Table 8B for the Kiechle samples. The odds ratio was calculated as described in Example 2.

TABLE 8A Reference AF AF Odds SNP ID F case F control p-value Ratio 2001449 G = 0.685 G = 0.777 0.005 1.59 C = 0.315 C = 0.223

TABLE 8B Reference AF AF Odds SNP ID F case F control p-value Ratio 2001449 G = 0.754 G = 0.716 0.267 0.82 C = 0.246 C = 0.284

The absence of a statistically significant association in the replication cohort should not be interpreted as minimizing the value of the original finding. There are many reasons why a biologically derived association identified in a sample from one population would not replicate in a sample from another population. The most important reason is differences in population history. Due to bottlenecks and founder effects, there may be common disease predisposing alleles present in one population that are relatively rare in another, leading to a lack of association in the candidate region. Also, because common diseases such as breast cancer are the result of susceptibilities in many genes and many environmental risk factors, differences in population-specific genetic and environmental backgrounds could mask the effects of a biologically relevant allele. For these and other reasons, statistically strong results in the original, discovery sample that did not replicate in the replication sample may be further evaluated in additional replication cohorts and experimental systems.

Example 4 Mode of Inheritance

To further describe the role of the SNP in breast cancer susceptibility the penetrance was estimated in both the discovery samples and the replication samples to allow inference of the mode of inheritance. The penetrance, defined as the probability of disease given each SNP genotype, was estimated from the case and control genotype frequencies, which provide estimates of the probability of each SNP genotype given the disease. Using Bayes theorem and an assumed age-matched population prevalence of breast cancer (all patients and breast cancer survivors) of 0.028, calculated from NCI data, results reported in Table 9 were obtained.

TABLE 9 Probability of Breast Cancer Penetrance Penetrance Based on Genotype in Discovery in Replication P(BC|GG) 0.022 0.022 P(BC|GC) 0.035 0.035 P(BC|CC) 0.048 0.048

These penetrances suggest that breast cancer susceptibility at this SNP is additive. These penetrances further suggest that breast cancer susceptibility at this SNP is inherited as a dominant trait.

Example 5 KIAA0861 Proximal SNPs

It has been discovered that a polymorphic variation (rs2001449) in a gene encoding KIAA0861 is associated with the occurrence of breast cancer (see Examples 1 and 2). Subsequently, SNPs proximal to the incident SNP (rs2001449) were identified and allelotyped in breast cancer sample sets and control sample sets as described in Examples 1 and 2. A total of seventy-five allelic variants located within or nearby the KIAA0861 gene were identified and fifty-seven allelic variants were allelotyped. The polymorphic variants are set forth in Table 10. The chromosome position provided in column four of Table 10 is based on Genome “Build 34” of NCBI's GenBank.

TABLE 10 Position in SEQ ID Chromosome Allele Genome Deduced dbSNP rs# NO: 1 Chromosome Position Variants Letter Iupac 3811728 246 3 184201246 t/c c Y 3811729 393 3 184201393 a/g a R 602646 628 3 184201628 c/g c S 488277 7586 3 184208586 t/c c Y 1629673 9223 3 184210223 a/g g R 670232 9933 3 184210933 a/t a W 575326 10154 3 184211154 t/c c Y 575386 10175 3 184211175 c/g c S 684846 10877 3 184211877 t/c c Y 471365 10907 3 184211907 g/c g S 496251 11289 3 184212289 g/a a R 831246 11793 3 184212793 t/c t Y 831247 11813 3 184212813 g/c g S KIAA0861-AA 13507 3 184214507 c/g g S 512071 14249 3 184215249 c/t c Y 1502761 14586 3 184215586 a/c a M 681516 14647 3 184215647 c/t t Y 683302 15004 3 184216004 c/t t Y 619424 16573 3 184217573 t/g g K 620722 16811 3 184217811 a/g a R 529055 18921 3 184219921 a/g a R 664010 19651 3 184220651 t/g g K 678454 20565 3 184221565 c/t c Y 2653845 25239 3 184226239 g/a a R 472795 25721 3 184226721 g/a a R 507079 27133 3 184228133 g/a g R 534333 27778 3 184228778 t/c t Y 535298 27906 3 184228906 t/c t Y 536213 28000 3 184229000 g/a a R 831245 30005 3 184231005 a/g g R 639690 30520 3 184231520 t/c c Y 684174 32195 3 184233195 t/c c Y 571761 32439 3 184233439 c/g c S 1983421 33858 3 184234858 t/c t Y 4630966 41716 3 184242716 c/t t Y 2314415 42450 3 184243450 t/g c M 6788196 43554 3 184244554 g/a g R 2103062 44211 3 184245211 a/g g R 9827084 44775 3 184245775 g/c c S 9864865 44962 3 184245962 a/g a R 6804951 45317 3 184246317 c/t t Y 6770548 45712 3 184246712 a/g a R 1403452 45941 3 184246941 t/c c Y 7609994 46520 3 184247520 g/t t K 9838250 47175 3 184248175 c/t c Y 9863404 48045 3 184249045 g/t t K 903950 48636 3 184249636 c/a t K 6787284 48689 3 184249689 g/a g R 2017340 48704 3 184249704 a/g c Y 2001449 48849 3 184249849 g/c g S 1317288 48850 3 184249850 g/a g R 7635891 49931 3 184250931 t/g g K 10704581 51510 3 184252510 —/tt t N 11371910 51526 3 184252526 —/a c N 10937118 51758 3 184252758 a/g a R 7642053 51975 3 184252975 c/g g S 3821522 53475 3 184254475 a/g c Y 2029926 55524 3 184256524 t/c g R 1390831 56754 3 184257754 t/g a M 7643890 57473 3 184258473 a/g g R 11925606 57497 3 184258497 a/c c M 9826325 57613 3 184258613 g/a a R 6800429 58023 3 184259023 g/a g R 6803368 58821 3 184259821 t/c c Y 1353566 59644 3 184260644 c/a g K 2272115 66217 3 184267217 g/a a R 2272116 66344 3 184267344 g/a g R 3732603 67326 3 184268326 g/c c S 940055 69777 3 184270777 a/c a M 2314730 83594 3 184284594 a/g g R 2030578 84579 3 184285579 g/c g S 2049280 85623 3 184286623 c/t t Y 3732602 126831 3 184327831 c/t a R 2293203 137878 3 184338878 a/t t W 7639705 147455 3 184348455 g/t t K

Assay for Verifying and Allelotyping SNPs

The methods used to verify and allelotype the seventy-five proximal SNPs of Table 10 are the same methods described in Examples 1 and 2 herein. The PCR primers and extend primers used in these assays are provided in Table 11 and Table 12, respectively.

TABLE 11 Forward PCR primer Reverse PCR primer dbSNP rs# (SEQ ID NOS 10-81) (SEQ ID NOS 82-153) 3811728 ACGTTGGATGACGTGTCGGTCCCCTTTCAT ACGTTGGATGACGCGCCACACCTCCCTAC 3811729 ACGTTGGATGTGGGCGAGGTTCTGCAGCGT ACGTTGGATGGTTTCGTTTCTCCGGCACAG  602646 ACGTTGGATGGAGGAGACCCAGGGTATGAG ACGTTGGATGTCTGGGACCGTTTACCGCA  488277 ACGTTGGATGCACACATTCTTCTCAAGTGC ACGTTGGATGGGAGGGACACAATTTAACTC 1629673 ACGTTGGATGGGCACCATGTGTGGCTAATT ACGTTGGATGAAGGATCACGTGAAGTCAGG  670232 ACGTTGGATGGAAGGTGGAGCAGACATTAG ACGTTGGATGACCTTAGTTATACCAGGCAC  575326 ACGTTGGATGACAGAGAGGCTTGGTCATAC ACGTTGGATGGGTGCTTGGTTGTGATTCTC  575386 ACGTTGGATGATTCCTGCAGGTACTGTGTC ACGTTGGATGTGAGCCCAAAACTACTGCTG  684846 ACGTTGGATGACCACCAGATAAAATCCCTC ACGTTGGATGAAGTTCCTCTGGTGGACAAC  471365 ACGTTGGATGTGAGTGACATTTGTGTCACC ACGTTGGATGCGGAGGATCTGAACAACTTC  496251 ACGTTGGATGGGGAGTCATTCCAATACCAG ACGTTGGATGGGAGTGAAAGGTCATATTGG  831246 ACGTTGGATGCACAATCTGTTAGAATGGTGG ACGTTGGATGCGTCAAGACTGAATGCATAG  831247 ACGTTGGATGGAAAATATAGTCCTACACAA ACGTTGGATGCGTCAAGACTGAATGCATAG KIAA0861-AA ACGTTGGATGGTTCTAATGTCACCCCTTCC ACGTTGGATGCAATGTGGCAAATTCTCTGG  512071 ACGTTGGATGCAAATCACCCCTGACAATTC ACGTTGGATGACCAGCACACTCAGCTTTAG 1502761 ACGTTGGATGCAGAAATATGAAGGTGGCCC ACGTTGGATGACCTTGAGCTCTGAGCCCTT  681516 ACGTTGGATGCTCCTCCTCAGAGGACTAAC ACGTTGGATGAGCCCAAGGACTCATACAAC  683302 ACGTTGGATGAAACATGGCGAAACCCGGTC ACGTTGGATGACCACGCCTGGCTAATTTTG  619424 ACGTTGGATGACCGGGAGCTCCCAGTCTG ACGTTGGATGTGGGAATCGGTTGAGAGCCG  620722 ACGTTGGATGGCAGCAAAGAATTGCCCGGC ACGTTGGATGTAAGGCGCCTGCAGAGGCGA  529055 ACGTTGGATGCTGCAGTTATCTGGGTGAGC ACGTTGGATGCCAGAACGTGGCTTGTTGGG  664010 ACGTTGGATGTGGTACCTCCAGGTAAAATG ACGTTGGATGTCCAGGCAGTCATTTTACCC  678454 ACGTTGGATGTTCTCTGCGGAGGAAAGTGC ACGTTGGATGTTAAGCCAGTCCCCACAAGG 2653845 ACGTTGGATGATCACTTGGACTCAGGAAGC ACGTTGGATGAGTCTTGCTCTGTTTCCAGG  472795 ACGTTGGATGTCACCTGAGCATCAGACATG ACGTTGGATGATAGTGGAAGGAGAAACGGG  507079 ACGTTGGATGAAGCCTCAGATGAGGCATAC ACGTTGGATGTCTGAAAGGGTTCAGGAAGG  534333 ACGTTGGATGCGTTGATGCACTGAAGGGAG ACGTTGGATGAGAGGCTAAATGTTGGCAGG  535298 ACGTTGGATGCAATTGCTCAGACCTTCACC ACGTTGGATGAATGCTAGAGACATTGCACC  536213 ACGTTGGATGTGAGGACCTCATTATTGGTG ACGTTGGATGCTGAGCAATCGAACTGCTAC  831245 ACGTTGGATGCTAGAATTACAGGTGCACAC ACGTTGGATGGCCAAGATGGTGAAACCTTG  639690 ACGTTGGATGGCATTTTACCACCATGTGGTT ACGTTGGATGCCTTCATGTTAATTCTGCCC  684174 ACGTTGGATGCTTTACTGAGTGGGCAAACG ACGTTGGATGTCTAAGTGGAACTCAGCAGC  571761 ACGTTGGATGAATATCCTAGGCTAGCAGTG ACGTTGGATGGTGCATAAATACATGAATAG 1983421 ACGTTGGATGTCCAGGTGTTATGGAGTCAG ACGTTGGATGGGCTTCTTGTGCTGCTGTGT 4630966 ACGTTGGATGTCAACAAAGATGCCAAGACC ACGTTGGATGGTGGATATCCATTGTCCTAG 2314415 ACGTTGGATGGGCTGAGTAACAGTCCATTG ACGTTGGATGCTTACAGTATCCAAAAAGGG 6788196 ACGTTGGATGTCAAAGGTAGGTTACCCCTG ACGTTGGATGATCCCCAATTTGCACATCCC 2103062 ACGTTGGATGTGCAGCCCTCAACCTTTCAG ACGTTGGATGCCTTATTCAGTTACTATTACG 9827084 ACGTTGGATGAAACACACACACCCACATAC ACGTTGGATGGGGAGAAAGAAAACAAAGGC 9864865 ACGTTGGATGCAATGCCTGCACTTAGACAC ACGTTGGATGAGTGATGAGAACATGGGCTG 6804951 ACGTTGGATGGCAATAGGACTCCCTTTACC ACGTTGGATGAAGATACGAATGGAGCCTGG 6770548 ACGTTGGATGTTTTTGAGCTTCACTGAGCG ACGTTGGATGCGTATCTCTAGCTCAAGCAT 1403452 ACGTTGGATGCAGAAGTTAGGATGCAGATG ACGTTGGATGCCAGTAGAGATAGAATTTTGG 7609994 ACGTTGGATGATACCTAGAGTTTGCCCAAC ACGTTGGATGAGCTGAGATCAATCCCTATG 9838250 ACGTTGGATGGTGGCAGTCAAAACACAGTC ACGTTGGATGACAGAGTAAGACTCCGTCTC 9863404 ACGTTGGATGGCTATTAGAAAGTCAGAGCC ACGTTGGATGTGTTCCAGAAGGTGTAGAAG  903950 ACGTTGGATGCTTCAGTTCAGGGAGAGATC ACGTTGGATGATAGGGCCCCCAGCATAAAA 6787284 ACGTTGGATGGCTTTCCCCTAAAGCATCTC ACGTTGGATGGATCTCTCCCTGAACTGAAG 2017340 ACGTTGGATGTATTCCACTGCCTGCTTTCC ACGTTGGATGGAAAACAGGAGGAAGTGGTG 2001449 ACGTTGGATGATGTCAAGTGCACCCACATG ACGTTGGATGAGGAAGAAACTGACGGAAGG 1317288 ACGTTGGATGATGTCAAGTGCACCCACATG ACGTTGGATGAGGAAGAAACTGACGGAAGG 7635891 ACGTTGGATGTCTCACCTTGCCTTTGGACG ACGTTGGATGCTGATGTCGCAAGGAACCAC 10704581  ACGTTGGATGCCTGTTGAATTATGGAGGAG ACGTTGGATGCTCTTCTTTCCATGGATCTTC 11371910  ACGTTGGATGCTCTTCTTTCCATGGATCTTC ACGTTGGATGCAGCTAATTTCTCCTGACAG 10937118  ACGTTGGATGATGCAAACTGGCTGGGAATG ACGTTGGATGGAGGAGGCTGTGAGAAAAGA 7642053 ACGTTGGATGATGCCCTGGATTGACCTAAC ACGTTGGATGGGGTTAGGGTGTGTATAAGG 3821522 ACGTTGGATGAACCCGCACTACAAGATTCC ACGTTGGATGGTCAGTCCCACATTCAGAAC 2029926 ACGTTGGATGTCCCGAACATAAAGACTCAG ACGTTGGATGGGTTGTAATTGGAACATTGG 1390831 ACGTTGGATGGTCTGCCAAAGTTCCCTTAG ACGTTGGATGAGGAAAGGGAAGAGAAACCG 7643890 ACGTTGGATGGACTGTGAGTTATAGGATAC ACGTTGGATGATGGGTCGGAGGATTTATAG 11925606  ACGTTGGATGCTCGGCTAAGGTACTCAATA ACGTTGGATGAGACCACCAAGTAAAATTGC 9826325 ACGTTGGATGTTGGGTTAATGCAGGGTCTG ACGTTGGATGCTAGTTCACCTGGGTCTATC 6800429 ACGTTGGATGCCAAAGCCCATGTTTTAAAAA ACGTTGGATGGTTTTTCTAAAATATGGGCT 6803368 ACGTTGGATGAAACCAGCTCAGGCCATTAC ACGTTGGATGATGCAAAATAAGCTCTGCCC 1353566 ACGTTGGATGGGTGTACTCTGCCATTTGTC ACGTTGGATGTGGAGGAGGTTCTAGTACCC 2272115 ACGTTGGATGAGTTGTGAGTGATTTCAGGG ACGTTGGATGCAGGCCTTCTTGCTCTTATC 2272116 ACGTTGGATGCTGTGCCTTCTGAGTAGTTC ACGTTGGATGATCTGTTGCCTTAGGTTCAC 3732603 ACGTTGGATGCTCTCAATTCCATCAGTCTC ACGTTGGATGCTTTACGAATTTCACAACAGG  940055 ACGTTGGATGTATGCTTCCAGTCTCTGACC ACGTTGGATGATAGGTAATCCAGTTGGGCC 2314730 ACGTTGGATGCTCAGGTAATCTGCCTTCTC ACGTTGGATGCAGGGATAATGAGAACAAATC 2030578 ACGTTGGATGAACAACCTTACTTCATGCCC ACGTTGGATGTTCTCCACTTTCTGGTCAAC 2049280 ACGTTGGATGTGGATACTGAGGGTCAACTG ACGTTGGATGCTTCCCAACATTTTCGGCTC

TABLE 12 Extend Primer dbSNP rs# (SEQ ID NOS: 154-225) Term Mix 3811728  GTCCCCTTTCATCTAAAC ACT 3811729  TCTGCAGCGTGCGGCGA ACT 602646 CCAGGGTATGAGCGGAGGA ACT 488277 AGTGCACACAGAACATTTAACA ACT 1629673  TGTGGAGACAAGGTCTCACT ACT 670232 TGGGCAAACAAGCCCAT CGT 575326 TGGTCATACCCTTCAAG ACT 575386 GAAGGGTATGACCAAGC ACT 684846 AGTTGTTCAGATCCTCC ACT 471365 TCCAAAACCACCAGATAAAATC ACT 496251 GTATTGTCCTCCAGTGA ACG 831246 AGAATGGTGGTGTATTTTTAC ACT 831247 TAGTCCTACACAATCTGTTA ACT KIAA0861-AA GGTATCAGGAAGAGTCA ACT 512071 CCCTGACAATTCCAAAACTAA ACG 1502761  GGAGGAGGCACTATTAAT ACT  681516 GGCCACCTTCATATTTC ACG  683302 CAGGAGATCCAGACCATCC ACG  619424 TGCGGCCCCCGCCGGGTT ACT  620722 GAATTGCCCGGCTCCGAAT ACT  529055 GAGCAGGCAGCACAAGT ACT  664010 ACCTCCAGGTAAAATGATTAGTT ACT  678454 CAGGGATGGTAATTGAC ACG 2653845 AAGCGGAGGTTGCAGTGAGC ACG  472795 GACATGTCCCTCTCGGCCT ACG  507079 GGCAATGTTTGCCCTTT ACG  534333 GGGAGAAAGTAACAGGGTC ACT  535298 CAGGTGGATGGGGACAC ACT  536213 TGGTGTTAAGTGGCGTG ACG  831245 CACACCACCACGCCCGGCT ACT  639690 CTGCTATTCATTTGTGTAGA ACT  684174 CTCTGATGTTACCTCCTCC ACT  571761 CTAGGCTAGCAGTGGGGTTG ACT 1983421 GGCAGGGAAGAGAAGAGC ACT 4630966 AGATGCCAAGACCATTCAAAG ACG 2314415 TAGTTGATGAAGATTTGGG ACT 6788196 AGGTTACCCCTGCTGACTTT ACG 2103062 GAGATCATTTCTCCTTCAAC ACT 9827084 CCACACCCATATATATTTATGCT ACT 9864865 AAAGATACACCGTTGAGAAGG ACT 6804951 GACTCCCTTTACCTTCATGG ACG 6770548 CTTCACTGAGCGTGGTGCC ACT 1403452 CACAGATGCTCATGGGTCC ACT 7609994 GTTTGCCCAACATATAAACAATAA CGT 9838250 AACACAGTCAAAATTTTGCTTCA ACG 9863404 GAGCCAAGTTTACATCAAGTTTA CGT  903950 AGATCACATTGCCAACCCCCA CGT 6787284 CCCGTCTCCTGCTGGTCA ACG 2017340 CCCTAAAGCATCTCACAGCCCC ACT 2001449 CACATGCCTGCTCGCCCCC ACT 1317288 CACATGCCTGCTCGCCCC ACG 7635891 TGCCTTTGGACGTCTAGCC ACT 10704581  TATGGAGGAGTAGATATTGGAA CGT 11371910  GAAAATTCCAATATCTACTCCTC CGT 10937118  CTGGGAATGAAATTAGGGCAG ACT 7642053 GTTCCCTTGACTTTCCTCAG ACT 3821522 GCATCTTCAGGAATCTTG ACT 2029926 CATAAAGACTCAGCATTCAGC ACT 1390831 GGTTAGGAAGAAATCTGTG ACT 7643890 ATCTAGATAATAAAGACCACCAA ACT 11925606  CTATTAATGGTGTTTGTCTATGG ACT 9826325 TAATGCAGGGTCTGCTGGAT ACG 6800429 ATCTCTAAGATATAACACTCTAC ACG 6803368 GTGCCTGCAAAGAAAGGAAC ACT 1353566 TTGTCAGTTATGAGACCTTG CGT 2272115 ATACCTCAGAATACAGCTTTTTTT ACG 2272116 TCTCATTTCTCCTCTCTTTC ACG 3732603 CTCATTTCCACCCTTCT ACT  940055 GTCTCTGACCACTTGACCCA ACT 2314730 TCCTTCTTCTCTGCTTT ACT 2030578 TCATGCCCATTGGGTTAG ACT 2049280 GGGTCAACTGTACCAAG ACG

Genetic Analysis of Allelotyping Results

Allelotyping results are shown for cases and controls in Table 13. The allele frequency for the A2 allele is noted in the fifth and sixth columns for breast cancer pools and control pools, respectively, where “AF” is allele frequency. SNPs with blank allele frequencies were untyped (“not AT”).

TABLE 13 Position in Breast Cancer SEQ ID Chromosome A1/A2 Associated dbSNP rs# NO: 1 Position Allele Case AF Control AF p-Value OR Allele 3811728 246 184201246 T/C T = 0.002 T = 0.003 0.952 1.28 C C = 0.998 C = 0.997 3811729 393 184201393 A/G A = 0.968 A = 0.947 0.268 0.61 A G = 0.032 G = 0.053 602646 628 184201628 C/G C = C = 0.344 G = G = 0.656 488277 7586 184208586 T/C T = 0.9 T = 0.898 0.92 0.98 T C = 0.100 C = 0.102 1629673 9223 184210223 A/G A = 0.93 A = 0.911 0.459 0.78 A G = 0.070 G = 0.089 670232 9933 184210933 A/T A = 0.138 A = 0.137 0.951 0.99 A T = 0.862 T = 0.863 575326 10154 184211154 T/C T = 0.876 T = 0.869 0.753 0.94 T C = 0.124 C = 0.131 575386 10175 184211175 C/G C = 0.224 C = 0.221 0.921 0.98 C G = 0.776 G = 0.779 684846 10877 184211877 T/C T = 0.202 T = C = 0.798 C = 471365 10907 184211907 G/C G = 0.258 G = 0.262 0.88 1.02 C C = 0.742 C = 0.738 496251 11289 184212289 G/A G = 0.841 G = 0.839 0.967 0.99 G A = 0.159 A = 0.161 831246 11793 184212793 T/C T = 0.229 T = 0.203 0.373 0.86 T C = 0.771 C = 0.797 831247 11813 184212813 G/C G = 0.17 G = 0.178 0.755 1.06 C C = 0.830 C = 0.822 KIAA0861- 13507 184214507 C/G C = 0.745 C = 0.762 0.557 1.10 G AA G = 0.255 G = 0.238 512071 14249 184215249 C/T C = 0.391 C = 0.363 0.376 0.89 C T = 0.609 T = 0.637 1502761 14586 184215586 A/C A = 0.417 A = 0.409 0.799 0.97 A C = 0.583 C = 0.591 681516 14647 184215647 C/T C = 0.762 C = 0.817 0.0906 1.39 T T = 0.238 T = 0.183 683302 15004 184216004 C/T C = 0.729 C = T = 0.271 T = 619424 16573 184217573 T/G T = 0.925 T = 0.929 0.812 1.06 G G = 0.075 G = 0.071 620722 16811 184217811 A/G T = A = 0.181 G = G = 0.819 529055 18921 184219921 A/G A = 0.398 A = 0.364 0.325 0.86 A G = 0.602 G = 0.636 664010 19651 184220651 T/G T = 0.549 T = 0.607 0.145 1.27 G G = 0.451 G = 0.393 678454 20565 184221565 C/T C = 1.000 C = 0.985 0.0998 0.00 C T = 0.000 T = 0.015 2653845 25239 184226239 G/A G = 0.825 G = 0.827 0.94 1.01 A A = 0.175 A = 0.173 472795 25721 184226721 G/A G = 0.921 G = 0.921 0.983 0.99 G A = 0.079 A = 0.079 507079 27133 184228133 G/A G = 0.166 G = 0.167 0.979 1.00 A A = 0.834 A = 0.833 534333 27778 184228778 T/C T = 0.502 T = 0.491 0.73 0.96 T C = 0.498 C = 0.509 535298 27906 184228906 T/C T = 0.275 T = 0.228 0.127 0.78 T C = 0.725 C = 0.772 536213 28000 184229000 G/A G = 0.726 G = 0.717 0.781 0.96 G A = 0.274 A = 0.283 831245 30005 184231005 A/G A = 0.979 A = 0.981 0.843 1.12 G G = 0.021 G = 0.019 639690 30520 184231520 T/C T = 0.882 T = 0.892 0.65 1.10 C C = 0.118 C = 0.108 684174 32195 184233195 T/C T = 0.698 T = 0.708 0.756 1.05 C C = 0.302 C = 0.292 571761 32439 184233439 C/G C = 0.601 C = 0.576 0.499 0.90 C G = 0.399 G = 0.424 1983421 33858 184234858 T/C T = 0.566 T = 0.58 0.669 1.06 C C = 0.434 C = 0.420 4630966 41716 184242716 C/T C = 0.359 C = 0.271 0.00247 0.66 C T = 0.641 T = 0.729 2314415 42450 184243450 T/G T = 0.974 T = 0.951 0.124 0.53 T G = 0.026 G = 0.049 6788196 43554 184244554 G/A G = 1.000 G = 1.000 0.967 0.00 G A = 0 A = 0.000 2103062 44211 184245211 A/G A = 0.674 A = 0.642 0.381 0.87 A G = 0.326 G = 0.358 9827084 44775 184245775 G/C G = 0.966 G = 0.928 0.0403 0.46 G C = 0.034 C = 0.072 9864865 44962 184245962 A/G A = 0.106 A = 0.185 0.000529 1.93 G G = 0.894 G = 0.815 6804951 45317 184246317 C/T C = 0.96 C = 0.904 0.00573 0.40 C T = 0.040 T = 0.096 6770548 45712 184246712 A/G A = 0.062 A = 0.159 1.12E−05 2.86 G G = 0.938 G = 0.841 1403452 45941 184246941 T/C T = 0.97 T = 0.932 0.0144 0.43 T C = 0.030 C = 0.068 7609994 46520 184247520 G/T G = 0.001 G = 0.002 0.918 2.34 T T = 0.999 T = 0.998 9838250 47175 184248175 C/T C = 0.52 C = 0.524 0.909 1.01 T T = 0.480 T = 0.476 9863404 48045 184249045 G/T G = 0.001 G = 0.002 0.887 2.58 T T = 0.999 T = 0.998 903950 48636 184249636 C/A C = 0.417 C = 0.406 0.739 0.96 C A = 0.583 A = 0.594 6787284 48689 184249689 G/A G = 0.475 G = 0.501 0.416 1.11 A A = 0.525 A = 0.499 2017340 48704 184249704 A/G A = 0.965 A = 0.945 0.195 0.63 A G = 0.035 G = 0.055 2001449 48849 184249849 G/C G = 0.738 G = 0.797 0.0285 1.39 C C = 0.262 C = 0.203 1317288 48850 184249850 G/A G = 1.000 G = 1.000 0.967 0.51 G A = 0.000 A = 0.000 7635891 49931 184250931 T/G T = 0.973 T = 0.947 0.121 0.49 T G = 0.027 G = 0.053 10704581 51510 184252510 —/TT — = 0.998 — = 0.997 0.949 0.83 — TT = 0.002 TT = 0.003 11371910 51526 184252526 —/A — = 1.000 — = 1.000 0.977 0.00 — A = 0 A = 0.000 10937118 51758 184252758 A/G A = 0.495 A = 0.51 0.629 1.06 G G = 0.505 C = 0.490 7642053 51975 184252975 C/G C = 0.002 C = 0.003 0.908 1.85 G G = 0.998 G = 0.997 3821522 53475 184254475 A/G A = 0.504 A = 0.52 0.62 1.07 G G = 0.496 G = 0.480 2029926 55524 184256524 T/C T = 0.001 T = 0.001 0.975 2.52 C C = 0.999 C = 0.999 1390831 56754 184257754 T/G T = 0.057 T = 0.076 0.284 1.36 G G = 0.943 G = 0.924 7643890 57473 184258473 A/G A = 0.001 A = 0.002 0.934 2.30 G G = 0.999 G = 0.998 11925606 57497 184258497 A/C A = 0 A = 0.001 0.956 C C = 1.000 C = 0.999 9826325 57613 184258613 G/A G = 0.002 G = 0.003 0.887 1.85 A A = 0.998 A = 0.997 6800429 58023 184259023 G/A G = 0.605 G = 0.59 0.662 0.94 G A = 0.395 A = 0.410 6803368 58821 184259821 T/C T = 0.002 T = 0.001 0.885 0.20 T C = 0.998 C = 0.999 1353566 59644 184260644 C/A C = 0.452 C = 0.469 0.604 1.07 A A = 0.548 A = 0.531 2272115 66217 184267217 G/A G = 0.673 G = 0.634 0.224 0.84 G A = 0.327 A = 0.366 2272116 66344 184267344 G/A G = 0.999 G = 1.000 0.876 8.14 A A = 0.001 A = 0.000 3732603 67326 184268326 G/C G = 0.773 G = 0.792 0.495 1.11 C C = 0.227 C = 0.208 940055 69777 184270777 A/C A = 0.778 A = 0.803 0.356 1.17 C C = 0.222 C = 0.197 2314730 83594 184284594 A/G A = 0.352 A = 0.311 0.183 0.83 A G = 0.648 G = 0.689 2030578 84579 184285579 G/C G = 1.000 G = 1.000 0.963 0.33 G C = 0.000 C = 0.000 2049280 85623 184286623 C/T C = 0.001 C = 0.005 0.576 30.94 T T = 0.999 T = 0.995

FIG. 1 shows the proximal SNPs in and around the KIAA0861 gene for females. As indicated, some of the SNPs were untyped. The position of each SNP on the chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the control group. The minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in FIG. 1 can be determined by consulting Table 13. By proceeding down the Table from top to bottom and across the graphs from left to right the allele frequency associated with each symbol shown can be determined.

To aid the interpretation, multiple lines have been added to the graph. The broken horizontal lines are drawn at two common significance levels, 0.05 and 0.01. The vertical broken lines are drawn every 20 kb to assist in the interpretation of distances between SNPs. Two other lines are drawn to expose linear trends in the association of SNPs to the disease. The light gray line (or generally bottom-most curve) is a nonlinear smoother through the data points on the graph using a local polynomial regression method (W. S. Cleveland, E. Grosse and W. M. Shyu (1992) Local regression models. Chapter 8 of Statistical Models in S eds J. M. Chambers and T. J. Hastie, Wadsworth & Brooks/Cole.). The black line provides a local test for excess statistical significance to identify regions of association. This was created by use of a 10 kb sliding window with 1 kb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p-values that were less than 110-8 were truncated at that value.

Finally, the gene or genes present in the loci region of the proximal SNPs as annotated by Locus Link (http address: www.ncbi.nlm.nih.gov/LocusLink/) are provided on the graph. The exons and introns of the genes in the covered region are plotted below each graph at the appropriate chromosomal positions. The gene boundary is indicated by the broken horizontal line. The exon positions are shown as thick, unbroken bars. An arrow is place at the 3′ end of each gene to show the direction of transcription.

Additional Genotyping

A total of fourteen SNPs, including the incident SNP, were genotyped in the discovery cohort. The discovery cohort is described in Example 1. Four of the SNPs are non-synonomous, coding SNPs. Two of the SNPs (rs2001449 and rs6804951) were found to be significantly associated with breast cancer with a p-value of 0.001 and 0.007, respectively. See Table 16.

The methods used to verify and genotype the five proximal SNPs of Table 16 are the same methods described in Examples 1 and 2 herein. The PCR primers and extend primers used in these assays are provided in Table 14 and Table 15, respectively.

TABLE 14 Forward PCR primer Revee PCR primer dbSNP # (SEQ ID NOS 226-239) (SEQ ID NOS 240-253) 7639705 ACGTTGGATGTGTCAGAAAGCAAACCTGGC ACGTTGGATGTTACAGGCATTGGAGACAGC 2293203 ACGTTGGATGCTGCATAATGGTGGCTTTGG ACGTTGGATGTGTGGGTGTTCACTTTGCAG 3732602 ACGTTGGATGCCCTCTTGTCAGGAAGTTCT ACGTTGGATGGAGACAGAGTTGAACTCCCG 2001449 ACGTTGGATGAGGAAGAAACTGACGGAAGG ACGTTGGATGATGTCAAGTGCACCCACATG 6804951 ACGTTGGATGAAGATACGAATGGAGCCTGG ACGTTGGATGGCAATAGGACTCCCTTTACC 3821522 ACGTTGGATGCGCACTACAAGATTCCAAGC ACGTTGGATGTCAGTCCCACATTCAGAACC 2293203 ACGTTGGATGTGTGGGTGTTCACTTTGCAG ACGTTGGATGCTGCATAATGGTGGCTTTGG 3811729 ACGTTGGATGTGGGCGAGGTTCTGCAGCGT ACGTTGGATGGTTTCGTTTCTCCGGCACAG  534333 ACGTTGGATGGATGCACTGAAGGGAGAAAG ACGTTGGATGAGAGGCTAAATGTTGGCAGG  575326 ACGTTGGATGTGAGCCCAAAACTACTGCTG ACGTTGGATGATTCCTGCAGGTACTGTGTC 2272115 ACGTTGGATGCAGGCCTTCTTGCTCTTATC ACGTTGGATGAGTTGTGAGTGATTTCAGGG  940055 ACGTTGGATGTATGCTTCCAGTCTCTGACC ACGTTGGATGGATAGGTAATCCAGTTGGGC 2017340 ACGTTGGATGGATCTCTCCCTGAACTGAAG ACGTTGGATGGCTTTCCCCTAAAGCATCTC  571761 ACGTTGGATGAATATCCTAGGCTAGCAGTG ACGTTGGATGGTGCATAAATACATGAATAG

TABLE 15 Extend Primer dbSNP # (SEQ ID NOS 254-267) Term Mix 7639705 TGATGCACGTGGAGCAG CGT 2293203 GCCCCTGGAAAAGGCCC CGT 3732602 GGAAGATGATGAGACTAAAT ACG 2001449 CACATGCCTGCTCGCCCCC ACT 6804951 TCCCTTTACCTTCATGG ACG 3821522 GCATCTTCAGGAATCTTG ACT 2293203 GCCCCTGGAAAAGGCCC CGT 3811729 GGTTCTGCAGCGTGCGGCGA ACT  534333 GAAGGGAGAAAGTAACAGGGTC ACT  575326 TGGTCATACCCTTCAAG ACT 2272115 ATCTTCTACACATTGATTCAG ACT  940055 TCTCTGACCACTTGACCCA ACT 2017340 TGGTGACCAGCAGGAGA ACG  571761 GGCTAGCAGTGGGGTTG ACT

Table 16, below, shows the case and control allele frequencies along with the p-values for all of the SNPs genotyped. The disease associated allele of column 4 is in bold and the disease associated amino acid of column 5 is also in bold. The chromosome positions provided correspond to NCBI's Build 34. The amino acid change positions provided in column 5 correspond to KIAA0861 polypeptide sequence of SEQ ID NO: 4. The corresponding amino acid position in the alternative KIAA0861 polypeptide sequence (SEQ ID NO: 5) can be easily calculated by adding 83 amino acids to the positions provided in column 5.

TABLE 16 Genotpying Results Breast Position in Amino Cancer SEQ ID Chromosome Alleles Acid AF F Odds Associated dbSNP rs# NO: 1 Position (A1/A2) Change AF F case control p-value Ratio Allele 3811729 393 184201393 A/G A = 0.917 A = 0.948 0.0542 1.65 G G = 0.083 G = 0.052 575326 10154 184211154 T/C T = 0.897 T = 0.885 0.545 0.88 T C = 0.103 C = 0.115 534333 27778 184228778 T/C T = 0.254 T = 0.249 0.85 0.97 T C = 0.746 C = 0.751 571761 32439 184233439 C/G C = 0.487 C = 0.465 0.492 0.92 C G = 0.513 G = 0.535 6804951 45317 184246317 C/T A819T C = 0.956 C = 0.915 0.007 2.02 C T = 0.044 T = 0.085 2017340 48704 184249704 G/A G = 0.027 G = 0.042 0.203 1.57 A A = 0.973 A = 0.958 2001449 48849 184249849 G/C G = 0.693 G = 0.782 0.001 1.59 C C = 0.307 C = 0.218 3821522 53475 184254475 A/G A = 0.372 A = 0.391 0.539 1.08 G G = 0.628 G = 0.609 2272115 66217 184267217 A/G A = 0.407 A = 0.444 0.246 1.16 G G = 0.593 G = 0.556 940055 69777 184270777 A/C A = 0.702 A = 0.753 0.0721 1.29 C C = 0.298 C = 0.247 3732602 126831 184327831 C/T S506F C = 0.008 C = 0.012 0.597 1.41 T T = 0.992 T = 0.988 2293203 137878 184338878 A/T L295Q A = 0.012 A = 0.015 0.690 1.24 T T = 0.988 T = 0.985 7639705 147455 184348455 G/T I276L G = 0.195 G = 0.189 0.794 1.04 G T = 0.805 T = 0.811

Example 6 KIAA0861 Expression Profile

A cumulative mRNA expression profile was determined for KIAA0861 using a panel of 56 cells and tissues that represent a plurality of cells from different human tissue types. Specifically, RT-PCR was performed in cDNA made from 56 cell lines and 11 normal tissue samples using the following primers: forward, CCAGTCGAAATGGACTTGAG (SEQ ID NO: 268); and reverse, CGCCTTCACAGTCTTCAAAG (SEQ ID NO: 269). The cDNA samples represent a variety of tissue types throughout the human body. The PCR reactions were done in a final volume of 10 μl using Hotstar Taq™ from Qiagen, Inc. Half of the PCR reaction was loaded on a 2% agarose gel to resolve the resulting product. From the expression profiling described above, KIAA0861 expression was found to be ubiquitous across several tissues, including small intestine, bladder, prostate and colon, for example.

Expression Pattern in Breast Cancer Cell Lines vs Normal Breast Tissue

Quantitative RT-PCR hME was used to measure relative levels of KIAA0861 mRNA in 4 breast cancer cell lines and 2 normal breast tissue cDNA. A 56 Mix is a cDNA mixture from 56 different cell lines representing the major human tissues was used as a positive control. The amount of cDNA used for each reaction was normalized based on expression of a house keeping gene, HMBS. KIAA0861 expressed significantly in MCF7 and MDA-MB-231 cell lines (both breast cancer cell lines), but not significantly in normal breast tissue.

Example 7 Inhibition of KIAA0861 Gene Expression by Transfection of Specific siRNAs

RNAi-based gene inhibition was selected as a rapid way to inhibit expression of KIAA0861 in cultured cells. siRNA reagents were selectively designed to target KIAA0861. Algorithms useful for designing siRNA molecules specific for KIAA0861 are disclosed at the http address www.dharmacon.com. siRNA molecules up to 21 nucleotides in length were utilized. Table 17 summarizes the features of two duplexes that were used in the assays described herein. A non-homologous siRNA reagent (siGL2 control) was used as a negative control.

TABLE 17 siRNA siRNA Target Sequence Specificity SEQ ID NO: siGEF1 KIAA0861 GAGACAAGTGGAGCTCCGT 270 siGEF2 KIAA0861 GTGGAGCTCCGTAAAGGCA 271 siGEF3 KIAA0861 ATCACCGCACTGCCATCGA 272 siGEF4 KIAA0861 GCATGCTATCCACGGAAGA 273 SiGEF1 STABLE KIAA-861 GAGACAAGTGGAGCTCCGT 274 siGL2 control Non-homologous CGTACGCGGAATACTTCGA 275 scrambled control

The siRNAs were transfected in cell lines MCF-7 and T-47D using Lipofectamine™ 2000 reagent from Invitrogen, Corp. 2.5 μg or 5.0 μg of siRNA was mixed with 6.25 μl or 12.5 μl lipofectamine, respectively, and the mixture was added to cells grown in 6-well plates. Their inhibitory effects on KIAA0861 gene expression were confirmed by precision expression analysis by MassARRAY (quantitativeRT-PCR hME), which was performed on RNA prepared from the transfected cells. See Chunming D. and Cantor C. PNAS 100(6):3059-3064 (2003). KIAA0861 gene expression was also determined by flow cytometric analysis of cells stained with a polyclonal chicken antibody specific for KIAA0861. A 50% reduction of KIAA0861 protein was seen in siGEF1-treated cells. Cell viability was measured at 1, 2, 4 and 6 days post-transfection. Absorbance values were normalized relative to Day 1. RNA was extracted with Trizole reagent as recommended by the manufacturer (Invitrogen, Corp.) followed by cDNA synthesis using SuperScript™ reverse transcriptase.

Strong inhibition of cell proliferation of MCF-7 breast cancer cells by siGEF1 was obtained. siGEF1 also strongly inhibited proliferation of another breast cancer cell line, T47D. These effects were consistent in all six experiments performed. Each data point is an average of 3 wells of a 96-well plate normalized to values obtained from day 1 post transfection. The specificity of the active siRNAs was confirmed with a control siRNA, siGL2, which is not homologous to any human sequences.

Long term inhibition of gene expression is desirable in certain cases. Therefore, included herein are embodiments directed to siRNA duplexes described herein (see Tables 31-36) that are less susceptible to degradation. An example of a modification that decreases susceptibility to degradation is in siSTABLE RNA described at the http address www.dharmacon.com. A stable version of siGEF1 was used in an invasion assay described above, except that the cells were replated 14 days after transfection. Inhibition of MDA-MB-231 cell invasion by siGEF1-STABLE was still seen 15 days after transfection. In contrast, the inhibitory effect of the standard version of siGEF1 was no longer apparent at this time and is comparable to that of the control siGL2-treated cells.

siRNA—Starvation Growth Assay

An aliquot of MCF-7 and T47D cells was plated on Boyden chambers with 8 μm pore membranes that are coated with growth-factor reduced matrigel (Becton Dickinson). In addition to growth factors, matrigel contains basement membrane components such as collagens, laminin, and proteoglycans, making it a more physiological growth surface for these breast cell lines. One day after transfection, cells were trypsinized and resuspended in media without serum and plated on top of the matrigel-coated membrane, which is suspended over media containing 5% serum. Cells were allowed to grow for 6 days then fixed in 2% glutaraldehyde and stained with 0.2% crystal violet. Evidence showed that under low serum conditions and on a physiological surface, inhibition of KIAA0861 expression by siGEF1 dramatically inhibits growth of two breast cancer cell lines by 95%. This effect is greater on the matrigel surface than on plastic at a high serum concentration where 50-60% inhibition in proliferation was seen.

siRNA—Invasion Assay

In addition to high proliferative rates, some cancer cells also develop the ability to metastasize. The metastatic potential of tumor cells can be assessed in vitro using Boyden chambers. MCF-7 and T47D cells are not metastatic and therefore do not traverse through the matrigel. For this assay, another cell line was used, MDA-MB-231, which is known to be highly metastatic. Cells in 6-well plates were transfected with 2.5 μg of either siGEF1 or siGL2 as described above. Cells were replated 5 days after transfection on matrigel-coated Boyden chambers suspended on media containing 10% serum. Cells were stained with crystal violet 20 hrs later and photographed. Cells that remain on top of the membrane were scrubbed off and the cells that had invaded through the matrigel and grew on the bottom of the membrane were photographed. Significant inhibition of MDA-MB-231 breast cancer cell invasion by siGEF1 was observed. Duplicate chambers were used in a Wst-1 assay to determine total cell number for both treatments.

Example 8 In Vitro Production of KIAA40861 Polypeptides

KIAA0861 Cloning

KIAA0861 cDNA was cloned into a pET28a (Novagen) and pcDNA3.1 vectors (Invitrogen) using a directional cloning method. A KIAA0861 cDNA insert was prepared using PCR with forward and reverse primers having 5′ restriction site tags (in frame) and 5-6 additional nucleotides in addition to 3′ gene-specific portions, the latter of which is typically about twenty to about twenty-five base pairs in length. A Sal I restriction site was introduced by the forward primer and a Sma I restriction site was introduced by the reverse primer. The ends of KIAA0861 PCR products were cut with the corresponding restriction enzymes (e.g., Sal I and Sma I) and the products were gel-purified. The pIVEX 2.3-MCS vector was linearized using the same restriction enzymes, and the fragment with the correct sized fragment was isolated by gel-purification. Purified KIAA0861 PCR product was ligated into the linearized pIVEX 2.3-MCS vector and E. coli cells were transformed for plasmid amplification. The newly constructed expression vector was verified by restriction mapping and used for protein production.

KIAA0861 DH/PH, DH and PH sequences were cloned out of a human brain library and subsequently cloned into pET28a (Novagen) for bacterial expression and pcDNA3.1 vectors (Invitrogen) for mammalian expression and encode a polypeptide domain described herein. In both cases, a directional cloning method was used and the sequences were verified (for use in NIH-3T3 primary focus forming assay and soft agar assay). The table below summarizes the different plasmid constructs.

TABLE 18 EXPRESSION CLONED VECTOR GENE REGION TYPE CLONING VECTOR KIAA0861 DH and PH bacterial pET28a NcoI/SalI sites domains KIAA0861 DH only bacterial pET28a NcoI/SalI sites KIAA0861 PH only bacterial pET28a NcoI/SalI sites KIAA0861 DH and PH mammalian pCDNA3.1 EcoRI/XbaI domains sites KIAA0861 DH only mammalian pCDNA3.1 EcoRI/XbaI sites KIAA0861 PH only mammalian pCDNA3.1 EcoRI/XbaI sites KIAA0861 full length mammalian pCDNA3.1 EcoRI/XbaI ORF sites DBS DH and PH bacterial pET28a NcoI/SalI sites domains DBS DH and PH mammalian pCDNA3.1 EcoRI/XbaI domains sites

Any method well-known in the art may be used to clone and express a target gene. For example, KIAA0861 cDNA may be cloned into a pIVEX 2.3-MCS vector (Roche Biochem) using a directional cloning method as described above. A KIAA0861 cDNA insert is prepared using PCR with forward and reverse primers having 5′ restriction site tags (in frame) and 5-6 additional nucleotides in addition to 3′ gene-specific portions, the latter of which is typically about twenty to about twenty-five base pairs in length. A Sal I restriction site is introduced by the forward primer and a Sma I restriction site is introduced by the reverse primer. The ends of KIAA0861 PCR products are cut with the corresponding restriction enzymes and the products are gel-purified. The pIVEX 2.3-MCS vector is linearized using the same restriction enzymes, and the fragment with the correct sized fragment is isolated by gel-purification. Purified KIAA0861 PCR product is ligated into the linearized pIVEX 2.3-MCS vector and E. coli cells transformed for plasmid amplification. The newly constructed expression vector is verified by restriction mapping and used for protein production.

E. coli lysate is reconstituted with 0.25 ml of Reconstitution Buffer, the Reaction Mix is reconstituted with 0.8 ml of Reconstitution Buffer; the Feeding Mix is reconstituted with 10.5 ml of Reconstitution Buffer; and the Energy Mix is reconstituted with 0.6 ml of Reconstitution Buffer. 0.5 ml of the Energy Mix was added to the Feeding Mix to obtain the Feeding Solution. 0.75 ml of Reaction Mix, 501 of Energy Mix, and 10 μg of the KIAA0861 template DNA is added to the E. coli lysate.

Using the reaction device (Roche Biochem), 1 ml of the Reaction Solution is loaded into the reaction compartment. The reaction device is turned upside-down and 10 ml of the Feeding Solution is loaded into the feeding compartment. All lids are closed and the reaction device is loaded into the RTS500 instrument. The instrument is run at 30° C. for 24 hours with a stir bar speed of 150 rpm. The pIVEX 2.3 MCS vector includes a nucleotide sequence that encodes six consecutive histidine amino acids on the C-terminal end of the KIAA0861 polypeptide for the purpose of protein purification. KIAA0861 polypeptide is purified by contacting the contents of reaction device with resin modified with Ni²⁺ ions. KIAA0861 polypeptide is eluted from the resin with a solution containing free Ni²⁺ ions.

Example 9 Cellular Production of KIAA40861 Polypeptides

KIAA0861 nucleic acids are cloned into DNA plasmids having phage recombination cites and KIAA0861 polypeptides and polypeptide variants are expressed therefrom in a variety of host cells. Alpha-phage genomic DNA contains short sequences known as attP sites, and E. coli genomic DNA contains unique, short sequences known as attB sites. These regions share homology, allowing for integration of phage DNA into E. coli via directional, site-specific recombination using the phage protein Int and the E. coli protein IHF. Integration produces two new att sites, L and R, which flank the inserted prophage DNA. Phage excision from E. coli genomic DNA can also be accomplished using these two proteins with the addition of a second phage protein, Xis. DNA vectors have been produced where the integration/excision process is modified to allow for the directional integration or excision of a target DNA fragment into a backbone vector in a rapid in vitro reaction (Gateway™ Technology (Invitrogen, Inc.)).

A first step is to transfer the KIAA0861 nucleic acid insert into a shuttle vector that contains affL sites surrounding the negative selection gene, ccdB (e.g. pENTER vector, Invitrogen, Inc.). This transfer process is accomplished by digesting the KIAA0861 nucleic acid from a DNA vector used for sequencing, and to ligate it into the multicloning site of the shuttle vector, which will place it between the two attL sites while removing the negative selection gene ccdB. A second method is to amplify the KIAA0861 nucleic acid by the polymerase chain reaction (PCR) with primers containing attB sites. The amplified fragment then is integrated into the shuttle vector using Int and IHF. A third method is to utilize a topoisomerase-mediated process, in which the KIAA0861 nucleic acid is amplified via PCR using gene-specific primers with the 5′ upstream primer containing an additional CACC sequence (e.g., TOPO® expression kit (Invitrogen, Inc.)). In conjunction with Topoisomerase I, the PCR amplified fragment can be cloned into the shuttle vector via the attL sites in the correct orientation.

Once the KIAA0861 nucleic acid is transferred into the shuttle vector, it can be cloned into an expression vector having attR sites. Several vectors containing attR sites for expression of KIAA0861 polypeptide as a native polypeptide, N-fusion polypeptide, and C-fusion polypeptides are commercially available (e.g., pDEST (Invitrogen, Inc.)), and any vector can be converted into an expression vector for receiving a KIAA0861 nucleic acid from the shuttle vector by introducing an insert having an attR site flanked by an antibiotic resistant gene for selection using the standard methods described above. Transfer of the KIAA0861 nucleic acid from the shuttle vector is accomplished by directional recombination using Int, IHF, and Xis (LR clonase). Then the desired sequence can be transferred to an expression vector by carrying out a one hour incubation at room temperature with Int, IHF, and Xis, a ten minute incubation at 37° C. with proteinase K, transforming bacteria and allowing expression for one hour, and then plating on selective media. Generally, 90% cloning efficiency is achieved by this method. Examples of expression vectors are pDEST 14 bacterial expression vector with att7 promoter, pDEST 15 bacterial expression vector with a T7 promoter and a N-terminal GST tag, pDEST 17 bacterial vector with a T7 promoter and a N-terminal polyhistidine affinity tag, and pDEST 12.2 mammalian expression vector with a CMV promoter and neo resistance gene. These expression vectors or others like them are transformed or transfected into cells for expression of the KIAA0861 polypeptide or polypeptide variants. These expression vectors are often transfected, for example, into murine-transformed a adipocyte cell line 3T3-L1, (ATCC), human embryonic kidney cell line 293, and rat cardiomyocyte cell line H9C2.

Example 10 Transformation of Normal Cells

Plasmid constructs of KIAA0861 and DBS DHPH domains in pcDNA3.1 vector were transfected into NIH-3T3 cells to determine the potential of these genes to transform normal cells. The oncogenic potential of DBS has already been established (Whitehead, I., Kirk, H., and Kay, R. (1995) Oncogene 10:713-721) and was used here as a positive control. Five μg plasmid was transfected into NIH-3T3 cells grown in 25 mm² flasks using Lipofectamine 2000 (Invitrogen). Approximately 10,000 cells were replated 1 day after transfection into 100 mm² dishes in media containing 10% serum. Cells were allowed to grow and express the plasmids for 4 days then media was changed to contain 2% serum. After 7 days growth in low serum, cells were fixed then stained with crystal violet. The low number of colonies that grew in cells transfected with the vector alone compared to those transfected with either KIAA0861 or DBS DH-PH domains indicate that these genes are transforming. Cells plated at 1000/dish show no growth in the vector alone treatment compared to a substantial number of colonies in the KIAA0861 or DBS treatments (data not shown).

To determine if KIAA0861 is able to induce a metastatic phenotype, a population of NIH-3T3 cells transfected with the above plasmids were selected by growth under 400 μg/ml G418 (geniticin) over a period of 2 months. These cells were then used in an in vitro invasion assay described in Example 8. Evidence showed that KIAA0861 as well as DBS transformed non-metastatic NIH-3T3 cells into cells that are able to invade through a matrigel matrix.

Example 11 Guanine Nucleotide Exchange Assays

Fluorescence spectroscopic analysis of N-methylanthraniloyl(mant)-GTP incorporation into bacterially purified Rho GTPases was carried out with a tecan XFlour spectrometer at 20° C. Exchange reaction assay mixtures containing 20 mM Hepes (pH 7.5), 50 mM NaCl, 5 mM MgCl₂, and 2 μM relevant GTPase were prepared in a 200 ul volume in a 96-well plate. The relative fluorescence (λ_(ex)=370 nm, λ_(ex)=465_(+/−)35 nm) was monitored before and after addition of 200 nM bacterially expressed Histidine tag fusions of KIAA0861, Dbs DH-PH domain proteins, or BSA. KIAA0861 and DBS DH-PH domain proteins are active in exchanging guanine nucleotide from the GST-tag fusions of the GTPases, RhoA and Cdc42. Based on the slope of a straight line fitted through the data points, KIAA0861 was equally active on both RhoA and Cdc42, while Dbs was more active on Cdc42 than on RhoA in this in vitro assay.

An alternative nucleotide exchange assay may be used as well, as described below. Guanine nucleotide exchange assays may be performed in 2 ml reactions. Briefly, nucleotide exchange is monitored as the increase in relative fluorescence of the GTP analog mant-GTP upon binding G protein in a reaction buffer containing 20 mM Tris (pH 7.5), 50 mM NaCl, 10 mM MgCl₂, 1 mM dithiothreitol, 50 μg/ml bovine serum albumin, and 10% glycerol. Prior to the addition of GEF, a 1 μM concentration of the appropriate G protein is incubated with 200 nM mant-GTP at 20° C. in a thermostatted cuvette, and fluorescence is measured using a PerkinElmer Life Sciences LS-50B λ_(ex)=360 nm; λ_(em)=440 nm; slits=5/5 nm). After equilibration, 10 nM GEF or buffer (uncatalyzed trace) is added.

Test molecules are screened using one or both of these procedures to determine which of them inhibit the guanine nucleotide exchange function of KIAA0861 or a portion thereof. The top ranked inhibitors identified in these screening procedures then are tested in other processes described herein, to determine their effect on cell transformation by KIAA0861 and cell invasion, for example. Top ranked molecules that inhibit cell transformation and/or cell invasion are identified as candidate therapeutics and are administered to animals and humans to determine their safety and therapeutic efficacy on breast cancer.

Provided hereafter is a KIAA0861 genomic sequence (SEQ ID NO: 1). Polymorphic variants are designated in IUPAC format. The following nucleotide representations are used throughout the specification and figures: “A” or “a” is adenosine, adenine, or adenylic acid; “C” or “c” is cytidine, cytosine, or cytidylic acid; “G” or “g” is guanosine, guanine, or guanylic acid; “T” or “t” is thymidine, thymine, or thymidylic acid; and “I” or “i” is inosine, hypoxanthine, or inosinic acid. SNPs are designated by the following convention: “R” represents A or G, “M” represents A or C; “W” represents A or T; “Y” represents C or T; “S” represents C or G; “K” represents G or T; “V” represents A, C or G; “H” represents A, C, or T; “D” represents A, G, or T; “B” represents C, G, or T; and “N” represents A, G, C, or T.

>3: 184201001-184348700      1 aaagccaaga ctcccattcc taaaccctag ctcaggtctc catctcttaa atccgagtga     61 cctctacaaa ctctccctga gaaggtgtcc agaacccttt tggaagcgag ggacagtgtc    121 actgtctttg gggttgacac ctgctctgag taactcacgg aaaacaagtt ccagctggga    181 agcccttgga cgcgccacac ctccctaccc gcagcccgtc ctgtggcgcc cgggactcca    241 gagtgYgttt agatgaaagg ggaccgacac gtcagggcca ccgcgggaag cgctgagggc    301 cactcaccgg ccagggacgc gaagagcgcg gccgccgcgc tgagctgccg gggcatggtg    361 ggcgctgggc gaggttctgc agcgtgcggc gaRgtccggg caggccccga atcggtgcca    421 gagaaaccta cctgtgccgg agaaacgaaa ccacctgctt atgagaagca gccgaaaagc    481 ccgcccaggg ccgctgggcg gggagggaaa ctccgccggc cccctcctac ccctacggag    541 cagggagggg cggggactcg gcgcagccgc cggggcccgg gcctctggga ccgtttaccg    601 cacgcgcgtg gtcccggcag cgccggcstc ctccgctcat accctgggtc tcctcctttc    661 tttttctttt ctttttgaga cgaagtctcg ctctgtcgcc cagggtggag tgcagtggcg    721 cgatctcggc tcactacaac ctctgcctcc cgggttcaag cgattcttct gcctcagcct    781 cccgagtagc tgggattaca ggcatgcacc accacacccg gctaattttt gtatttttag    841 tagagacggg gtgtcaccat attggccagg ctggtctcga gctcctgacc tcgtgattcg    901 cccgcctcga cctcccaaag tgctgggatt atagacgtga gccaccgagc ccggccaggg    961 tctcctcttt tatttctttt ctttttattt cttttgtttt gttttgtttt gttttgtttt   1021 ttgagacaaa gtctcgctct gtcgccaggc tggagtgcag tggcgggatc tcggctcact   1081 gccctggttc aagcgattct cctgccgcag cctcccgagt agctggggtt acaggcgccc   1141 gccaccacgc ccagctaatt tttgtatttt agtagagacg gggtttcacc ctgttggcca   1201 ggctggtctc gatctcctga ccttgtgatc cgcccgcctc ggcctcccaa agtgttggga   1261 ttacaggcgt gagccactgc gcccggccca gggtctcctc ttttctaaca gctcgggtac   1321 ctttctggga acccagagac gcttctcagc cgggagaaag ccagccacta ggcgagcagg   1381 agcctaaaaa cccctaagca ccctgactcc atgtcttccc agggagtctg cggcagccgc   1441 gctccacgcc caggcctcgc caggaccgcg gtttgcggga agcaacagga gcacagccca   1501 gaggcgctag gtctggctgg gagctcgcgc tgccgactcc ccggcgtgcg gcgtcgggga   1561 acctctagga gccttggatt cttcagctgt aaaacggaca taataatgcc cactcccagt   1621 gtgttttttt attttctttt ttctttttct ttctttgttt ttgtttgttt gtttttgttt   1681 ttgtttttga gacagggtct cactctgtcg cccaggctgg agggcaatgg cgtgatctcg   1741 gctcactgca aacttgggtt caggcgattc tcctgcctca gcctccacag tagctgggat   1801 tacagatgtg cgccaccacg tccggctaat tttttgtatt tttagtagag accaggtttc   1861 accgtgttgg ccaagctggt ctcaaactcc tgaccccagg tgatccgccc gcctcggcct   1921 tccaaagatc tgggattaca agcgtgagcc actgtgcctg gccccaggtg gttttacaga   1981 ccagaaaatc ctggaacaaa aaacacacaa tatcgttttt tttttttttt tggagtcagg   2041 gtctcgctct atcacccagg ctggagtgca gtggcgtgat ctcggctcac tgcaacttcg   2101 acctcctggc ctcaagtgag tctcccacct tagcctcctg agtagctggg accaaaggcg   2161 cgtgccacca cgcccagcta ttttatttta ttttatgtag agaggaggtc tcgctgtgtt   2221 gcccaggctg gtctcgagtt cctggcctca aatgatcctc ctgcgttagc caaccattgg   2281 gattacaggc gtaagccacg gcccacggct caacaacgct gacaggcaac cttttaatgt   2341 cttatctcct tcctctatta attggattgt ctgtcaaaac aacgatgttt tgacagggct   2401 tgagtcccag tggggaatac acatttaagc agtatattag gagaccctcc ttatcactag   2461 attgagggct ttcagcctag cctcaaatta ttttctgaaa aataactttg gctacaacta   2521 ttttgtctta ctatgttgct ccaaacacta atcaagtaaa cttaaccaaa gcttgcagtg   2581 tgtttcagaa tggaattttt atggtgaaaa gtgagggtta acttgtgcca gtcaacctag   2641 tttcagcaac tacctgcttt ctgatctttg agacagttta ttcaaaagac gataattaag   2701 tgggtataga ctgtgtgcca ggcactcttc ttattccatt taagcgccat agccactcta   2761 tatggacact gttgttatta tcgctgcccc atttcgcaga tggagaaact aagcacaaag   2821 aagggagttg cccagagtca cttagataat aaataccgaa acctgaccat aaatcttgtc   2881 tgccttgaga gtctaggatt ttaagcacat agccgggcgc agtggctcac gcctgtaatc   2941 ccagcacttt aggaggccga ggcgggcgga tcacgaggtc aggagatgga gaccatcctg   3001 gctaacacag tgaaacccag ttctattaaa aatataaaaa aattagccag gcgtggtggc   3061 aggtgcatgt agtcccagct actcgggagg ctgaggcagg agaatggtgt gaacccagga   3121 ggtggagctt gcagtgagcg gagatcgcgc cactgcactc cagcccgggc gacagaagga   3181 gactccgtct caaaaaaaaa aaaataaata aaataaagct gctcctctta ccctggaaat   3241 tccaagggat ttaggagctc tgtttcagga accagggtca aagaccaagt attaaaacaa   3301 aagattctcc tagaactctg gcatataagg attttaggag ctctgtctta gaaactggga   3361 cagagaccaa agatatatta ttatatcgca gtatcatagt ttattatttt caaaaaacgt   3421 tttctggctg gtacagtggt tcatgcctat aatccaagca ctttgggagg ccaaggtggg   3481 agggtcactt gaggccagaa gttcaagtcc agcctgggca acacagggag accctgccac   3541 tattaaaaat tttttaaatt agctgggcat ggtggcacat gcctgtagtc ccagctactt   3601 gggaggctga agcaggagga ttgcttgagc ctgagaggtc aagactgtag tgagctgcga   3661 tcaagcgact gcactctagc ctgggtgaca aagccagacc ctgtgtctaa aaaaaagaaa   3721 agaagaggaa aaaaaaaggt ttttatttca actaagttgt tggatttatt agcataaagc   3781 ttttcataac aatcccttgt cttacaatat ctgtagtata ggtagtgatg tcacttcttt   3841 tattactaat attaataatt tgtattttct ctcttatttc cctgctaata ttaataattt   3901 gtattttctc tcttttttcc ctgataagtt tggctggagg ttgatcaatt ctattcatgt   3961 tttcaagaaa aaaaaaaaaa tttcatttca tcgagttcct tcattgttct tatgttttct   4021 gttttattca tttctacctg atctttatta ttttctttct tctacttaac ttgtgtttta   4081 tttgctcctc tttcaatagt tttcaaagat ggaacttgcc tgggatatcc cagcacttta   4141 ggaggctgag gtgggaggat cacctgaggt caggagttca agaccagcct ggccaacatg   4201 gtgaaactcc gtctctacta aaaatacaaa aattagctgg gtgtggtggt gggcacctgt   4261 gatcccagct actcgggagg ctgaggcagg agaatcgctt gaacccggga ggtggaggtt   4321 acagtgagct gagatcacgc cactgcactc cagcctgggt gacaggagct agactctgtc   4381 tcaaaaaaaa aaaaagaaaa aaaaaaagat ggaagttgag gccagtggta taaaatcttt   4441 cttcatctct aataaacagg tttactgtta tgaacgtccc tctaattact actttagttg   4501 gatcccacaa gtttaatatg ttttcatttt catataatta gaaagacttc ctaatttcct   4561 tttgctatct cctcgactca tggttattta aaatagcatt atttcatttc caaacatata   4621 gtttttcaga tatctttctt ttattgattt ttaattccac tgtggttgaa aacataatta   4681 tggacttaaa tcttacaaat ttattgatat ttgttttttg accaagacta tggttggtta   4741 cattagtttt caggactgac ataacaaagt gccacagact gggtaattaa accacagaca   4801 tttgctttct tataattctg gaaaccagac atgtgagatc aaagtgtcag ccgggttggt   4861 tttttctttt tccttttttt tttttttttt tgagacagag tctctttctg tcacccaggc   4921 tgggatgcag tggtgtgatc tcggcttact gcaaattctg cctcccaggc tcaagcgatt   4981 ctcctgcctc agcctcccga gtagctggga ttacaggtgc ctctcactgc acctggctaa   5041 tttttgtatt tttagtagag acggggtttc accatgttgg ccaggttgat ctcaaactcc   5101 tgatctcagg taatacaccc gcctcggcct cccaaagtac tgagattaca ggcgtgagcc   5161 actgcacccg gcccgggtta gttctttcta aggcctctct ccttggctag tagacacctt   5221 tgtttcacat ggtcatccct ctgtgcatgc ctttgtctgt cctaatctcc tcttcttata   5281 aggacattag tcaggtagga ttagtgccta ctctttgaac tcgttttacc tcttaaagac   5341 cctatctccg aatatagtca cattctgaga tacttggggt taagacttgt attagtccat   5401 tttcacgctg ctcataaaga catacctgag actgggaaga aaaagaggtt taattggaca   5461 attccacatg gctggggagg cctcagaatc atggtgggag gcgaaaggga ctttttacat   5521 ggtggcggca agagaaaatg aggaagaagc aaaagcagaa acccctgata gataagccca   5581 ccagatatca tgagatttat tcactgtcat gagaacagca cgggaaagac cagcccccat   5641 gaatacatta cctcttcctt ggtccccccc tccccacaat atgtggggat tctgggagat   5701 acaattaaaa ttgagatttg agtggggaca cagccaaacc atatcattct gtccctggtc   5761 cttccaaatc tcatgtcctc acatttcaaa accaatcatg cctttccaat agtccctcaa   5821 agtcttaact catttcagca ttaacctaaa agtccacagt ccaaagtctc atctgagaca   5881 aggccttccg cctatgagcc tgtacaatca aaagcaagct agttagttcc tagatacaat   5941 gggggtacag gtattgggta aataaagcca ttccaaatgg gagaaattgg ccaaaacaaa   6001 ggggttacag ggcccatgca agtctgaaat ccagtgaggg agtcaaattt taaagctcca   6061 aaatgatctc ctttgactcc aggtcttaca tccaggtcac gctaatgcaa aaggtaggtt   6121 tccatggtct tgggcagctc cacccctgtg gctttgcagg gtacagcctc cctccaggct   6181 gctttcatgg gctggtgttg agtgtctgca gcttttccag gcacccagtg caagctgtca   6241 gtggatctac cattctgggg tttggaggac aaaggccctc ttctcacagc tgcactaggc   6301 agtgccccga tagggactct gtgtgggggc tctgatccca catttccctt ctgcactgcc   6361 ctaagaggtt ctccttgagg gccccacagc ttccaccctc tgaaccatag cccaagctat   6421 gcattggccc ctttcagcca tggctggagc agctgggaca gagggcacca agtcactagg   6481 ctgcacacaa catggggacc ctgggcctgc cccacaaaac ccctttttcc tcctgggcct   6541 ccaagcctgt gatgggagag gctgctgtga aggtctctga catggccttg gagacatttc   6601 cccatggtct tggggattca cattaggctt cttgctactt atgcaaattt ctgcaaccag   6661 cttgaatttc tccccagaaa atgggttttt cttttctgtc acatagtccg gctgcaaatt   6721 ttccaaactt ttatgctctg cttcccttat aaaactgaac gcctttaata gcacccaaat   6781 cacctcttga atgttttgct gcttagaaat tttttccacc agatacccta aataatctct   6841 caagttcaaa gttccacaag tctctagggc aggggcaaaa tgtggccagt ctctttgcta   6901 aaacataaca agaggcacct ttgctccagt tcccaaaaag ttcctcatct ccatctgaga   6961 ccacctcagc ctggatctta ttgtccatat cactatcagc attttgggca aaaccattca   7021 acaagtctct aggaagttcc aaactttccc acattttcct gtcttcttct gagcccttca   7081 aactgttcca atctctgcct gttacccagt tccaaagttg ttccacattt tcaggtatct   7141 tcagcaacgt ttcactctac tggtagcaat ttactgtatt agtccatttt cacactgctg   7201 ataaagacat atctgagact gggaagaaaa ataggtttaa ttggacttac agttccacat   7261 ggctggggag gcctcagaat catggtgaga ggtgaaaggc acttcttacg tggtagtgac   7321 aagagaaaat gaggaagaag caaaagcgga aacccctgat aaatccatca gatctcatga   7381 gacttattca ctatcacgag aatagcatgg gaaagaccgg cccccatgat tcaattacct   7441 ccccctgggt ccctcccaca acacatggga attctgggag atacaattca agttgagatt   7501 tgggtgggga cacagccaaa ccatatcaag acttctacat atgaattttg gagggacaca   7561 atttaactca taatagtgga ctgtcYtgtt aaatgttctg tgtgcacttg agaagaatgt   7621 gtgtattctc tcattgttgg attcagtgac ctataaatgt taattaggtt aaactaattg   7681 atgtagggaa aagaaagaga gatcagactg tcactgtgtc tatgtagaaa gggaagacat   7741 aagagactcc attttgaaaa agacctgtac ttcaaacaat tgctttgctg agatgttaat   7801 ttgtagcttt gccccagcca ctttgcccca gccactttga cccaacttgg agctcacaaa   7861 aacatgtgtt gtataaaatc aaggtttaag ggatctaggg ctgtgcagga cgtgccttgt   7921 taacaaaatg tttacaagca gtatacttgg tcaaagtcat cgccattctc tagtctcaat   7981 aaaccagggg cacaatgcac tgcggaaagc tgcagggagc cctgcccttg gaagcggggt   8041 attgtccaag gtttctcccc atgtgacagt ctgaaatatg gcctcgtagg atgagaaaga   8101 cctgactgtc ccccagccca acacccataa agggtctgtg ctgaggtgga ttggtaaaag   8161 aggaaagcct cttacagttg agatagagga aggccactgt ctcctgcctg cccctgggaa   8221 ctgaatgtct tggtgtaaaa cccgattgta catttgttca actctgaaat aggagaaaag   8281 ctgccctgtg gtgggaggtg agacatgttt gcagtaatgc tgccttgtta ttctttactc   8341 cactgagatg tttgggtgga gagaaacata aatctggcct atgtgcacat ccaggcatag   8401 taccttccct tgaacttaat tatgatacag attcttttgc tcacatgttt tttgttgacc   8461 ttctccttat tatcaccctg ctgtcctact acattccttt ttgctgaaat aatgaaaata   8521 ataatcaata aaaactgagg gaactcagag gctggtgccg gtacaggtcc ttggtgtgct   8581 gagtgccggt cccctggact cactgttgtt tctttatact ttgtctctgt gtcttatttc   8641 ttttctccgg ctctcatccc acccgactag aaatacccac aggtgtggag gggcaggcca   8701 ccccttcaat tgatagtatg gttcaagaac aaatggtatc aacttaggat ggtttaactt   8761 atgatttttc aactttagaa tggtgtgaag tctgtatgca ttcagtagaa ggcatacttt   8821 gaatttttat cttttcccaa gctactgcta tgggacaaga tactctctca caattctggg   8881 cagtggcagc aagcctcagc ttccagtcag cacccaatcc caagggtaaa caactgatac   8941 agccattctg tttttcattt ttagcaaaat actcaataaa ttacatgagg cactcaatgc   9001 tttattataa gacaagcttt gtattagatg atttgcccaa ctgtaggcta atgtaggtgt   9061 tctgagcaca tttaaggtag actatactat gccatgatgt ttggaaggtt aggtaaattt   9121 aatgcatttt cgacttagaa tactttcagc ctcccaaata gctgggacca caggtgtgtg   9181 gcaccatgtg tggctaattt tttgtggaga caaggtctca ctRtgtcgcc caagttggtc   9241 tcaaaatcct gacttcacgt gatccttcca ctctggtctc ccaaagtgcg attacaggtg   9301 tgagtcacca cacctggccg tgttgtcaca cattttaatt ctttataaat tagaaacttc   9361 acaacacatt gttcttattt aaaatttaaa caattatctt taaatataca taatatataa   9421 atatgtatat aatatataaa tatatacaat atataaatat ataagatata ttatatatat   9481 aatatataaa tatataatat ataaatatat aatatgtaaa tatataatat ataaatatat   9541 gtaaatatgt aatatgtaaa tatgtaatat gtaaatatat aatatgtaaa tatataatat   9601 ataaatatac ataatatata aatatataat atataaatat ataatatata aatatataat   9661 atataaatat ataatatata aatatatatt atatataaat atataatata cataaatata   9721 tataatatat aaatatatat aatatacata aatatataat atataaatat atataatata   9781 tataaatata tatggggaaa aaagctttta tacttactca tgtgattacc gtttcttgca   9841 gtctttattc ctttgtttag atccttggga tttttgttgc tatggtgacc ttagttatac   9901 caggcacttc aaatcttacc ttgtgtttag gaWatgggct tgtttgccca aaggtcttcc   9961 ctaatgtctg ctccaccttc aactttaggt cttctctgct gtcagtttct ctctccatac  10021 acttgtagct ctcccaggag tattccatcg ttacttgtta gtcagtgctt attagcgggg  10081 tggtgggatc tgagaggtga ggtgcttggt tgtgattctc agttctgatt cctgcaggta  10141 ctgtgtccct gggYcttgaa gggtatgacc aagcStctct gtccctcccc gcagcagtag  10201 ttttgggctc agcacatatt cctgcccctt ccccagaatc agagggtttt ttgttgttgt  10261 tgttttgttt ttcaagtttt tgttcctttt tctccatctg tgttggattt accagccccc  10321 taggagcgtc agtatttgtt acccttcctc caggctttta aggcctttgt aggagagatg  10381 ggccaatagc atctgagcat ggttttgtgt ctttcttgta gcaactgata ttcctcaccc  10441 ccaggtctat gccaggaagg atgcttcctt acatgcctgt aatcccagct accggggagg  10501 ctgaagcaga agaatcactt gaacccagga ggcagaggtt gcagtgagcc aagatcatgc  10561 cactgcactc cagcctggca acagagcgag acttcattca aaaaaaaaaa aagctcttac  10621 actcttctgc atattctgca aagtatactc tgggaaggct ggtttagagg atctctaact  10681 tttctaatat ttcataatgc atggctgatg tttaacatga actcagactc ccatcagatt  10741 ttacagatct gggtctgctt ttagttctaa tgcttcagcc agggcacttt aatctgaggt  10801 cacagcattc ccaattgtca gcagtatttt gaaatctaag ttcctctggt ggacaacagg  10861 caacatcata tagccaYgga ggatctgaac aacttctaag tctgagSgat tttatctggt  10921 ggttttggat tcttttccat ttatacttaa acaaggtgac acaaatgtca ctcacagaaa  10981 agccactctt ttttcccctg tttttaagaa gtctaaaaat gcacaactat ttcctgaatg  11041 agcagtgtgt ccaggtgtag aatagggaat gtcgtgtcca ctgggaacag acggtgtacc  11101 tggcacaatt ttatgagaat tatcccatga ttcctgtccc accctgcagc ccaccatggg  11161 ctcagtgggg agtctgagtc tctgagcccc aaagggtagc tttttcccag acgacaccag  11221 caagcagagc agcagcttgg ggctttgaca aggaggctgg agtgaaaggt catattggac  11281 catcctggRt cactggagga caatacccca ggaacctcca gcaatgactg gtattggaat  11341 gactcccctg gtcacccacc tgtggcaaga catttgcaaa cagggtcttc ctatgaggct  11401 tgtgaggttt ggccaatttg cattctgcta gaattattaa gtatggacat cctcatagat  11461 ccagaaaagg gccgagatcc aaaggattga gaatgctttg gggggtgttt ccatagtgag  11521 tggctggcta ttcaacagtc aaatgtttga gaggatacaa gtgatgtgtt tcctgaggca  11581 agtggtcaga acccaacaga ctcttctgtt cagctaagat gagagaccat ctgaagttct  11641 tacatgttct cacagagaaa tggattctca tagggaaatg gatatattgt gatagacact  11701 gtaagcagag aagttgactg agaaacacac acacacacac acacacacac acacacgtca  11761 agactgaatg catagatgtg tattataaaa agYgtaaaaa tacaccacca ttStaacaga  11821 ttgtgtagga ctatattttc ttttttttta atttattatt attatacttt aagttttagg  11881 gtacatgtgc acaatgtgca ggttagttac atatgtatac atgtgccatg ctggtgtgct  11941 gcacccatta actcgtcatt tagcattagg tatatctcct aatgctatcc ctcccccctt  12001 cccccacccc acaacagtcc ccagaatggg atgttcccct tcctgtgtcc atgtgttctc  12061 attgttcaat tcccacctat gagtgagaat atgtggtgtt ttgttttttg tccttgcaat  12121 agtttactga gaatgatgat ttccaatttc atccatgtcc ctacaaagga catgaactca  12181 ccatttttta tggctgcata gtattccatg gtgtatatgt gccacatttt cttaatccag  12241 tctatcattg ttggacattt gggttggttc caagtctttg ctattgtgaa tagtgccaca  12301 ataaacatac gtgtgcatgt gtctttatag cagcatgatt tatagtcctt tgggtatata  12361 cccagtaatg ggatggctgg gtcaaatggt atttctagtt ctagatccct gaggaatcgc  12421 cacactgact tccacaatgg ttgaactagt ttacagtccc accaacagtg taaaagtgtt  12481 cctatttctc cacatcctct ccagcacctg ttgtttcctg actttttaat gattgccatt  12541 ctaactggtg tgagatggta tctcactgtg gttttgattt gcatttctct gatggccagt  12601 gatgatgagc attttttcat gtgttttttg gctgcatcag tgctctacac gttcagagaa  12661 acttctctag tgacgaacta tagaaatgat ccctgaaagt atagtcttag gactatattt  12721 tcttttgact tgggaggcat gtttattgct gttaatgctg caaagggctc tacgtgcttt  12781 aaaaaatccc aatctgttgc attcataagc ctgggttgga tctaaagcag cctcccactt  12841 ttggaaaggc atccccacga cctttccatg gttgctgaat gcagctggag gcagtcacag  12901 ctggtgatgt ccggagccca ttccccactg tgctggtctg cagaacttct gcatgccatt  12961 cccacaagca ggtctctgcc ctgctctcct ccacctccct tgtcagagga agtctgcact  13021 tcacagcttt ctggtctcaa ccctcctcca tccctacaga tgtgtaagca gcaggaatca  13081 aaaggtgaag gagagggggc aactcacctc cgatggacac gtgaaaagtg ggagatggat  13141 aaaatcaaga aggagcttaa gatatccaga aatgtaaact gtgtttggaa aagtaaggtc  13201 aggagaagca tgggactcct gaggttgctc cctactatct tgcagacttg ctgcaggacc  13261 aaatgaagca ggatctgtca agcaccaggg ccagctctta agcttagtgc ctttctgaac  13321 cctgtgaccc agcagcctcc atcaactcgt cctacctgcc atgcacagct cctctgtgcc  13381 cctgtacctg agctcatgct attccctctg ccaggatgcc cttctccttc tccaccagga  13441 gaagaacact tgccagtaag acccagttct aatgtcaccc cttcctgacg gtatcaggaa  13501 gagtcaStga tggtgtttta tgctcccaga gaatttgcca cattgtgttg tgattatttt  13561 tccacatctg tctcccccac tggaatgaga gcctcactca tcttcatacc tccctggtct  13621 ctacctggtg ccagaaccat cctcagggca ggggaatgct caggaaatag atattgaata  13681 aaataagtgt atccatccat ccatccatcc atccttccat ccatccagac atatatacat  13741 atgtaaacat tctgatggtc aaatggaaca atgtgggctg aagaataatg caggtagaag  13801 aacctaagat tacagattct tgatttggga ggaactttat tttattgtgc aaacagtcta  13861 caaatttgaa acatactcta tcaagaaaga cactgttgtt aggaaagggc gtgggagagg  13921 gtggccccac aaaacagtgt aaagttctaa agaatttgag aggaacactc tgcgggaccc  13981 tgttccaagg gcatctttct aagagtctgt cccttaggct ctgccccttt ttggccacgt  14041 tgtcaaaggc cttctttatg aacgaagtga gacacattat ttttgttttc ttgaattcta  14101 aagtgattgt cttgagttcc ctgggggaga ctcctgggag gtgtggccca ctctattcca  14161 gaaacaaacc aggagactga agactcatcc caatcccata tcccatgaaa atgtgaaatc  14221 aaatcacccc tgacaattcc aaaactaaYg gaatcctaac ataactctat gtaactctaa  14281 agctgagtgt gctggtaatt aagcttccag cccacccttc ccagctctgg ggtgcagggt  14341 caccatgggc tccttcctgt gtgcacccca ccccaccccc actgtcagca tcctgtctcc  14401 accctgagag tgacagctgg ttccagtagc gggagttggt tccagcttgc cattttccta  14461 gcactcctat aaccagcctg ctgatgccca ttcagagaca gcagcacggg ctggccatgt  14521 cccctctcca gaattctgcg tccagctcct ggaccttgag ctctgagccc ttgggccacg  14581 tgtacMatta atagtgcctc ctcctcagag gactaacccc cagccctagg gccaccttca  14641 tatttcYgag ttttgatatt ttcaacctct tttctttgtt gtatgagtcc ttgggctggg  14701 agcttgcagt caaaatcttc atgatatctc attatcacta ctttttttta aatctctact  14761 agctggataa caattattta tattaaattc tctcttgaaa taactgatac agtgtctctt  14821 gattgaaact tgactagtag actaagaatt ctaactctaa ataattctga gggccgggtg  14881 tggtggctca cacctgtaat cccagcactt tgggaggcca acgcgaatgg atcacctgag  14941 gtcaggagtt ggagaccagc ctggcaaaca tggcgaaacc cggtcaggag atccagacca  15001 tccYggctaa catggtgaaa cctcatcttt acttaaaaaa aaaaaaaaat acaaaacaaa  15061 attagccagg cgtggtggca gatgcctgta gtcccagcta ttcaggaggc tgagacagga  15121 gaatagcgtg aacccgggag gtggagcttg cagtgagccg agattgcacc actgcactcc  15181 agcctgggtg acagagagag actccgtctc aacaacaaca acaacaaatt agccgggcat  15241 ggtggcaggt gcctgtaatc tcaactactt gggaggctga ggtaggagaa ttgcttgaac  15301 ccgggaggcg gaggttgcag tgagccaaga ttgcggcact tcactccagc ctgggtgaca  15361 agagcaaaac tccatctcaa aaaaaaaaaa aaagaaaaaa aaatctgaca attaaataaa  15421 gaacagaaaa aaaatttgaa tggcaaatac aaagctgaaa agaaataact gagaataaat  15481 aactctgaaa atagctcaaa aactaaatac ctcaaaaact ctttaaaaat tcagaaaata  15541 taatgttcaa atatgaagta atgctgaaaa tgaaataact aaaaaccaag taacttgaaa  15601 aaaagaacat aaaaataaac aactcaaata taaaataact gaaaataaat acctgtgaac  15661 ataagcaact cgaaaaccag ataactaggg gaaacccttc attaaaacat ttcactctga  15721 aaataaataa cttgacagta gttcatgaac ttccagtgag tgtttaatag tcaaataagt  15781 tactgtaaaa ataaataact caaaaactcc aataagataa agtgaaataa ctatgaaggt  15841 aaataactca gataataatt gtaaagataa ttaaaaataa attccggctg ggcgcggtgg  15901 ctcacgcctg taatcccagc actttgggag gccgaggcag ggggatcacg aggtcaggag  15961 atcgagacca tcctggctaa cacggggaaa ccccgtctct actaaaaatc caaataaaaa  16021 attagccggg cgtggtggcg ggcgcctgta gtcccagcta ctcaggaggc tgaggcagga  16081 gaatggcgtg aacccgggag gcggtttgca gtgagccgag atcacaccac tgcactccag  16141 cctgggcgac agagcgagac tccgtctcga aaaataaata aataaataaa taaataaatt  16201 ccaagcaaat taatttgaac atctgtaact cctaaaacaa aacaactaca tataaataac  16261 tgaaaatgaa caactaaata actttgaaaa taaagtaact ataaaccagt aactgaaact  16321 ataaaattaa ataaccagaa agctacaaag aaatgaaaat caactaataa aactaaaaat  16381 aaaatacaac tgaaactaac aaaactcatt taaaaactaa taaatcgata cataaaatac  16441 ctcagaaagt aacttggaac gaactctctc tacaacgaag caatcttggc actaacacga  16501 acaccccatg aacgctcgca gggatgctgg gaggcggacc gggagctccc agtctgcggc  16561 ccccgccggg ttKgagcggc tccggctcct ccaaggctcg ggctaagcgg ctctcaaccg  16621 attcccaccc cgccctggag aaatgcgggc gtgtctgcag gcatttttga ttgtcacgat  16681 ttgggggcgg ggtggaggat ggggtggcgc attcctggca cctagtgggt agaggccagg  16741 ggtgctacta aacatcctac cgtgggagca cagagaagcc cacgcagcaa agaattgccc  16801 ggctccgaat Rtcgaagtgc gcggtcgaga aggcgtgggc tgcgggctct gctcgcctct  16861 gcaggcgcct tagagcagct ccgaggtccc ccgtgcggag ctaggcgcgc acccaggaca  16921 cccctcgggc tcctcggagg aggccctggt tgtccccttt ctgccgccgc cgaggctccg  16981 gctgctttct gcgtagctgg gcagggcccg ggcccccaca ccgcctctcc cgggaatgcg  17041 ggcgctctgg agccgaggag cgggggcgtc cgcagggagg tcagctctcc tgggcggagg  17101 tcctcgggcg cagcgccctc gcctggaaac cagccgtcgc ccccgcagga gccagccggc  17161 ccgtggacgc cccagcgcgc tcctcctcgg tgctgcgggt cgccctgcaa ttccgagaag  17221 aaagtcagag acgccgtggc ccaaagaggc gcttagtctt tcctcgctca cactcacgtt  17281 tcctcctcat cgcgttcttc tttttctccc tggctgcttt ctcccctctc caggaaagca  17341 gatttggagg aacaggtttc gtgactgtcg tccgactgga aaaggcccgc gagctggaag  17401 ggaggggacg ggtgcaccct cagagttatt gctggaggct gtggccagac cgggcaaggt  17461 ggtgactccc gctggcaggc tgaggcccac cccagccctc ccacctgggc cacggggctc  17521 tcagcgggag ccccagttat gaccggacac cagcgcaccg ccaaggagac agccacgtgg  17581 ggacatgctg gactaggagg gtcagagcca gtttgagggt ctggtgacct cggctccctg  17641 gcttaaccag gtccttatgg gtgagaatcc tgaggagggg gagagggatg gaggctaggg  17701 acaggaggca ggaggagctg atgaatagga aggagggaag aagatgaaag aaacaaaagg  17761 gaagtaatta cactcagagc actgctctcc ttttccatgt cttctgcgcc ttcacagtct  17821 tcaaaggtgt ccatggagct aaactccctg ctggagcagc cttagggaga gaaagggaag  17881 gatggggcct ctgtggggtg ggaggacatc ccctctgccc atggcagggt gtagcaggca  17941 gtgcctgttg caggcacggt cctccccatc tctaactcct gctctccaag ggcctgcact  18001 gcgctgggct gtgagggggt ctgtgatctc caggctgctt ttccagcgcc gagatgccgt  18061 aattcaccga gaaggcgcgt ccacatgctg ttcatggccc tacctccccg ttcctccaag  18121 aaaacagtca ttgttttttg tgtttgccag tcttctaacc acgcctcctt cccttcctcc  18181 tcccctgtct ctttctcacc ctcccctcct tgcctccttt tctctcccct aattaatgtc  18241 catttcccat ctccctggca gcctctgcca agtgtcactg ctccccataa gggaaaatca  18301 gaggaacaag caagtgcatc catcctgcct ctctctgcag tgaactgatt aattaatcca  18361 tcagtcttgt ctatggcgca catgttacat ccctggggcg gtgttggaca ctgtggggaa  18421 cagcagccac tgccaaatac tgaacaactg ccctgtgcct ggtggtatgt taggcatttg  18481 tcaaagttta agcctcacaa ccctgtaagg gtctcagccc cctttacagt tggggaaaca  18541 gacagcaatg gtcacttggc caagtcctct tggcctgtgg cagggcagct gtcttctcca  18601 gcactcctgc tctttaccct cgctctgagt gagatggagt ctctgcccca catggctcac  18661 aagccagggt agggagcaga gcatctgcag aaaggtccca acacaggaca gccccagacc  18721 gagggtcagc tgagtagtct acgcggcggg agccgtgcta ggaaagtgtc tgctcaggcg  18781 agaattcagg gaggtttggg caggacttga ggggcagaca ggactggaga gggagggcat  18841 tctgggcaga ggcatggcca gagggcggta ggcggcagtg gagggagctg cagttatctg  18901 ggtgagcagg cagcacaagt Rgcgtctcct gggctgctgc cccaagcccc caacaagcca  18961 cgttctgggc cccaggccct ccccagagca gatcagtggg ggctgtgtga gtaacatggg  19021 ggcggggggg cagctgggca gcacctccct ggaggcccct ctgaaatcct gcctgactct  19081 ggcaggctcc gagggggctg gacaccctcc tctcaggttg aagcaagtcc tggttgagtt  19141 cctagtccca ggaggtggga ggggcaaggg gtggagggca gaggagaaac tgcctcaggg  19201 atgtgccccc tgccttcatc ctccagacag gacttgggag catctaagga aacccaagac  19261 tcctctttag agaagtcatc cagccctggg gtccccttat gccaggagca agcagtgaga  19321 atggaagaat gattgtcttg ctgaaagttc tgtgatggag ggatagaggg acagagggag  19381 ccatgccctt gaccatcccc tgcatgaata ggaagggctg tgtctccagg gtccatggcc  19441 tctgtgcccc ggatgatgcc agggctgcta gggaccatag agccacccac tgggaggctg  19501 gcggttgggc ctggctcagg agccttcgtc agccataggc agccacagcc tggggtgggc  19561 agggctggga ggcgacacag gaactgaaaa acctgacaag ctctagcccc tccgcagggt  19621 aagtggtacc tccaggtaaa atgattagtt Kgttccagcc cctctgcagg gtaagtggca  19681 cctggggtaa aatgactgcc tggagctggc agctgctttc cctgctctcg cgggccctgc  19741 agggaagcgg ggaagggaag ggggcacagc gctgggcaca gaggggctct cagaccctgg  19801 actcaactgt ttcagggtca tctgaaacag tcaactgttt cctctagccc attccctgcc  19861 tccaggcgag gatttgcctg aacgtggaaa gaggaaggat cctcccagtg ctgtcaaccc  19921 cagattccac ctccctgtgg gggactgtca gcgcaggccc tgacaacgca gagaaagaca  19981 caggacccac ctgggccagt gacagcagga gctccgggtg ccacaggtga gggtggggat  20041 gcctggagca ccacgggggg cctggtttag tctagagcca ggttttccat acaccttaga  20101 gtgcaacctc agggagatgc aaattttacc ccctaacaca gcatacacgc agaaacacat  20161 ttatacaatt caaacacaag cggacggaac aatatttacc cttagagtgt gtgaagtcct  20221 catctgtccc acctcatcct atcatgcttt gttctatcct aggagacaaa gcaggagggg  20281 gggctgcgga ggtgggggag tctcatccaa gcccttgggt gacacgtctc tcctgagaca  20341 aactgcagct gctctgggtg tgccctcgcc tgtctccctc caggccccgg gttcctgcca  20401 gcagagacag taacctattc accaggtatc ccccagggct cctggaagaa actcagaatt  20461 ctcagaacca gaaaacctta gagagcatcc tgcaggcaaa gcccctggtt ctctgcggag  20521 gaaagtgcgg ctcacagggt gccccgccag ggatggtaat tgacYaccag gctgtgtgcc  20581 ttgtggggac tggcttaagg ccctgtggga gctgagtcag ggccaggacc ggggtgtcct  20641 gactcctaga gatcatgttc ccttcctcac ccaggccttc cagtcccagc cctgggcttt  20701 tatttattta ttttggagac agagtctggc ttgtcaccca ggttggagtg cagttgtgtg  20761 atcacggctc actgcagcct tgacttcctg ggttgaactg atcctcccac atcagcctcc  20821 tgagtagctg ggaccacagg cacatgccac cacacccagc taatttttgt attttttggt  20881 agagatgggg ttttaccatg ttggccaggc tggtcttgaa ctcctgagct caactgatct  20941 gcccacctca gcctcccaca gtgctgggat tacagatgtg agccatcatg cccacctcct  21001 gggctgactt ttgctgtctt acatcatctg catatttaat cccctgctgg attcactggt  21061 catgggctct gaggccctaa gagtcttagg cactaaggag ctggcagcac tgaggggacc  21121 ccaaaatctc agactcagga tctggccagt cacaggcatg tgagggaaca actgagaggc  21181 ccattgcccc atggcaggag aaggtgctct ggagtcagtc agacctgagg gcagtcagac  21241 ctgattctca ctctgtcact cactagctgt gtgatcttgg atacatcact taacctcttg  21301 agcatcagct tccttatctc taaaatggag ataataacat cgattttgca gtcttggtat  21361 gaggattagc aaatcttctg ataaagaaaa atgcctggta catcatagga attcaacaaa  21421 tagtacctgt tatgattatt gtgtatagca attacaataa tactaaagag agggtctcaa  21481 aacagctctg ggcactccag gtgtgctatt attacttaca tttcagggag gtttgtctgc  21541 cattgtctca tcctcataaa cactcaggga aagaaacatt ataaggataa taaatggctt  21601 taaaaagaaa cagagcaaac acacacacac acacacccct cagaaaaacc atgccaaaca  21661 cacaggctct tgacaaatat tcaatctgat tatagcaaaa ctgttttgtt ttgttttgtt  21721 tttttgtttg tttttgagac aggggtctcg ctctgtcgcc caggctggag tgcagtggcg  21781 tgatcttggc tcacagcaac ctccttctcc cgggttcaag caattctcct gtctcagcct  21841 ccagagtagc tgggactaca ggcacatgcc actatgcctg gctaattttt gtatttttag  21901 tagagacagg gtttcaccat attggtcagg ctggtctcga actcttgatc tcaggtgatc  21961 cacctgcctt ggcctcccaa agtgtgagat tacaggcgtg agccaccatg cccagctgat  22021 tatagcaaaa ttctaagtga tagttgtatt cttggaaaat gaatggaacg acttttgtcc  22081 cagccaagat ctagtggtgt gttggagcag atacaccctg agtctctggg gcactctcag  22141 tctatgtatc agataagcat aaggagtatt ggtggagaag gacaagaatg ggaaaggtgg  22201 gcgatgagaa ttcctgcaga taaggactgg tgagagtatt ctcttttgaa acccttagtc  22261 gacaatcttt ctggtgcata tcagataagc tgaatggttt aggaaatcta gtgttcacat  22321 ttagtgctta gaattctaag cttttttatt ttgcttaaac aaatggaatg aaatttatta  22381 acaagtgaac ctagtaatga gctgaaatta ttctcaccag catacatatt tttggtaaat  22441 tatagacttt gaagacaaaa tcatggtgtt tcccttactg tccagtggat ggcacaaaga  22501 gaccattgta gatcctgctg gttcagcggt agctctcaat ccatagatat taaatgggca  22561 aattctattt ttattgtctt tcaaactaga tttttctcaa tgcacaactt tttttttctt  22621 tttctttttt ttttttgaga cggagtctcg ctctattgcc aggctggagt gcagtgacac  22681 gatctcggct cactgcaacc tccgcctccc gggttcaagt gattctcctg cctcagcctc  22741 ctgagtagct ggggactaca ggcgcatgcc accatgccca gctaattttt ttgtaatatc  22801 agtagagaca gggtttcacc atgttggcca ggatggtctc gatctcctga ccacgtgatc  22861 cgcccacctt ggccttccaa agtgctggga ttacaggcgt aagccaccgc gcttggccaa  22921 ttatgcacaa ctttttaagg accatctcta tcacatgaac taaaggatat cattttcact  22981 tgggggtggg aagtggtgag ctgcttaaaa gcaatgccta aaccccctgg gctttccttc  23041 ctttcacttg gaagaaccag ggggtaacta actgctaaca attccatgct ttcagagatc  23101 taggcgcaag cctaagggct tcatctcatt taatcctcat ggcaagactg tacagtatta  23161 tctccatttt gtaaaatgaa tgataaaaag aacttaagca cagacaggct atagaatcca  23221 tccaaagaga tggagctgca cttgaggctg ggtctttgag acctgagttt gagcccttca  23281 tcattgtgtt gtgtgtgctg ctaaccaatt tcccctctct gtcttctagg atttaataca  23341 tggtgtcaca ttctgtctga tccatcccag tagctctcca gagctcccaa caggagagag  23401 ttccaaaatg tttccagggg tactaggctc ggttacaata ttttgcttgg tggcccagag  23461 tataaacgtg gatatcttag gctggtctat agctgaaacg tctatttcat ttgcaagcct  23521 atctttggct aagaggaagt gaatcattct tgagaacatc taattaattg ctttcagatt  23581 ccacatgttg acattctcag ggcacatttt ttttttcccg tgcatttctg ttgtcaaagt  23641 ccctgccagc tcctaaggca gtctgagctg gctgtcttag actttcagag ctgctggaag  23701 cttgggggag ggaggggctg taggtcaaag aaactttata acctagcttt acctcccagc  23761 tcagccacca gctgccctca aatgttctgg attggaataa gcccaaagat gagtggcagg  23821 agggaagggc aagccaatac gtctagtttg gttcagtcaa agccttgccc atttcatcag  23881 aattttaatg gaaaatttcc aataagatta aaatataagg tcaccccaat tttagtatgg  23941 ctccatttaa aaaaaatcat gcatatcttt gttttgcaat ggggccttac tcttgcccag  24001 gagaggtgcg gtggtacaat catggcttac ggcagcctca acctcctgag ctcaagcaat  24061 cctttcacct tggcctgcca aatagctagg attacagaca cccaccacca tgcccagcta  24121 atttttaaaa aaaatttttt gtaggtccag tgcagtggct catgcctgta aatctcagca  24181 ctttgggagg ccgaggcaag cggatcacct gaggtcagga gtttgagacc agcctggcca  24241 caatggtgaa actgccgtct ctactaaaaa cacaaaaatt agcgaggtgt ggtggtgggc  24301 acctgtaatc ccagctactc gggagactga agcaggagaa tcaattgaac ctgggaggtg  24361 gaggttgcag tgagccaagt tcgtgccatt gcactccagc ctgggcaaca agagtgaaac  24421 tccgtctcaa aagtttttgt ggagatgggg tcttgcgata ttgcccaggc tggtctcaag  24481 ctcctggata tcaagtgatc ctcctgcttt ggcttcccaa agtgttggga ttacaggcat  24541 gagccaccac aactgaccaa atcatgcatt tctatagaca acgtctgcaa gaagatatta  24601 atggtgatta tatctggtta tgttggattt gtatttttat ttgtactttc ctgtattttc  24661 taaattcctg acattctaca tgtgtactcc tttaataatc agaaaaggaa acttaaaata  24721 gttaaaacca attggtcaga tatgtaaaat aacccaccat ctctccagag agggctgttt  24781 gctagaactt attttcttca ttgaaatact agagtgcccc aataagtttg aataacacaa  24841 aaaaaaagat aatgaaagta actaaattat ctaggcccaa aaggaaatgc cacaaaaatt  24901 ggcaaagaaa caaccatgac gtgctatacc ggatagctcc taggccccct tggagaccct  24961 gaggtacccg acgagggacc tgtagtaagg ctggcagaca ggttcttcct ctgttagctc  25021 tgaggtacaa cagttattct catttttatg tctttcacat ggccagaact ttgcaaatca  25081 gaggcaaagt gaattcagaa ttaaaaattt tcagcaccat ccaagtcagt aaaacagtct  25141 tagcttataa cctttatttt ttttattatt ttattatttt tttatttttg gatggagtct  25201 tgctctgttt ccaggctgga gtgcagtggc atcatctcRg ctcactgcaa cctccgcttc  25261 ctgagtccaa gtgattctcc tgcctcagcc tcccgagtag ctgggacgac aggcatgcgc  25321 caccacgccc agccaatttt tgtattttta gtagagacgg ggtttcacca tgttggccaa  25381 gatggtctca atctcttgac ctcctgatcc tcccgcctcg gcctcccaaa gtgctgggat  25441 tacaggcgtg agccaccgcg cccagccctt tatttttact gtaagctgcc agtaaacagc  25501 acacccacct gtctgctgac tgtcccatct ggaagttgtg taggtcctta ctgaatctta  25561 ccttcttccc ttcccctacc cagaccccat cccagctggc acgtggaatc ctttcccttt  25621 ctcctcatgt caatagacca gcaggcaaag agagcagtta ccatagtgga aggagaaacg  25681 ggtcctagtc gtggtgagga ggtagggctg ctgcttaaca Raggccgaga gggacatgtc  25741 tgatgctcag gtgatccact aggacatctc gcaatactcc catgcccagc tgtaactatg  25801 atggccaagt ccaagaacca tagcctgata agagtctagg acccccacta tacatgccac  25861 cagtagaagt gcctgatggg gagagggatg tctagaatga gtaggaggga atgatgacaa  25921 agcacagcca aggaccaact gcagtggcag gtggcagcca cagattgagt taagtaggat  25981 tcttgctgtg ttccctagtg gaagcatctc ccttgttgag acccactgag gttaaagttg  26041 gagacatggg gagaaaacaa tgtccccgtg ttcatcacta gagtcatgat gggcaaggtc  26101 acaactcatt tgacttcttg gctcccagat tcatatactc tgcctcttgg gggcacttca  26161 ccataataca gccattggtt cagttgtatg ctgcatcctg agggacagca ctccatcccc  26221 gcagtgccat gtccaaactg ctgccccgcc tcactctcaa gaggctgtcc cactactctg  26281 tcaggcacca gcttctgaat gcagtggtaa gactctgtcc tgtgatcacg tctatggctg  26341 attctctttt gccatactga gtcccctgat caaagcagtg ttatacagcc tggcgcgctg  26401 gctcatgcct gtgatcccag cactctggga ggcagagggg gatggatcac tagaggtcag  26461 gagttcaaga ccagcctggt caacatggtg aaaccctgtc tcctctacta aaaattcaaa  26521 aattagctgg gtgtggcatg agcctgtaat cccagctact cggaaggctg aggcaggaga  26581 atcacttgca cccaggaggt ggaggttgca gtgatccctg atggtgccgt tgcactccag  26641 cctgagcgac aagtgtgaaa ctctatcgca aaaaaaaaaa gcaacgctta tgattgatca  26701 cacactgtac tttctaacct gaaggggtct ggtaaaatca acttgtcttc tcaagggaca  26761 gtaccaaact gggggctcag cactgacctc tgctggcatg tcggacattt aaaagtggta  26821 gcagctagag cagtcttgaa gacatggagc cctcactgct ggggccgagc actgctgggt  26881 tcaattctgc taccaaggct tctccatttc cgagcccatg gtgcaggcac tggggtgcct  26941 aagaggaagt gcactaactg gacttctgct atgcaccttc tttagggggc attaaagcac  27001 agttcaaaga ctctcacatt ttgagttaac ccccacaggc ctgtgtgccc cttctccagt  27061 caggtcatgt tgtcatttga tcctagttat tattctgaaa gggttcagga agggaggtgg  27121 ggacagatct tgRaaagggc aaacattgcc ttgtatgcct catctgaggc ttccgcatag  27181 tcttagggga ctgggaaccg tcttaagtga atctcacctt cttcccttcc cccagccagg  27241 ccctagccct actggcatga ggaatcctat ctctttgtcc tcatgccagg agaccagcag  27301 gcaaagagag cagttaccat actggaagga gaaatgggtc ctggtcatca gaaggaggta  27361 ctcctcaagg gacagtacct gagggagaca cttccactag ggaacacagc aagaatccta  27421 cttaactaaa tctacggctg ccgcctgctg ctacagttgg tccttggctg tactttgtag  27481 tacaaggcga agtaggccac tgctgccagc ccaggaggtc aagggcattt acccagacat  27541 cctcattccc agtttttggg tcccttatct tccctctcag gatctaatga ctcatctact  27601 ggcactagga gtccaaaatg accagatggc agactcgcct aggctgagaa ttcttccttc  27661 tctcaaattc tgccactttt accagcccag tctgcctctg ctgtaggaga tgagaggcta  27721 aatgttggca gggaggcccc ctgattctga tcctgtgttt taaatcgata attagtcYga  27781 ccctgttact ttctcccttc agtgcatcaa cgggaggctc ttagcaacag cctcccaact  27841 ctaccttcct gatagataat attttcctca aagctctcaa atgctagaga cattgcacca  27901 gctggYgtgt ccccatccac ctgtatctga tcccaggtca ccacaggtga aggtctgagc  27961 aatcgaactg ctacagcagg ccaggtacca ggactctcaR cacgccactt aacaccaata  28021 atgaggtcct catggccatt tggcctgcag tgagccaaca ccagaatccc atccattctg  28081 ggacccctcc tgctactaac ggtcttcagt caggttcccc agaagcagat tatgcaaatg  28141 tgttattgaa aaacggcttt cagatgaaac ctggaagaca gtgaaggccg aagctagggc  28201 agggaagaag ccaggcacag acgtgggctt agcagaagtc tagcatcagc ctgatcccct  28261 gggcagtact ggagcatgga tggcaccaca ggttaccatc ttgagacaga aggactggct  28321 tctgtaccct gaatcagtaa gccattggtg ggccataagc cacccttagg ggaggacata  28381 acctcacagg catttcctgg ctggacgacc ctgggcagct gagggcaact gcctgaaggg  28441 cacaacggtg agccattgtc agctaacctc acagcagcag agacatgggg ccaacagcct  28501 ataaagaggg tctgggcagg ctgtcaatac tctctactgt aatactgtat atgtggttta  28561 ttttaaaaac ttttttagaa ggctttattt tattttattt tttgagaagg agtttcgccc  28621 ttgttgccca ggctggagtg caatggcgcg atcttgactc accacaatct ctgcctcctg  28681 ggttcaagga attctcctgc ctcagcctcc cgagtagctg ggattacagg cgtgtaccac  28741 catgcccggc taattttttg tatttttagt ttcatcatgt tggcctggct agtcttcaac  28801 tcctgacctc aggtgatccg cccacctcgg cctcccaaag tgctgggatt acaggcgtga  28861 gccaccgcac ccggctgact ttattttttt agagcagttt taggttcaca gaaaaactga  28921 gaggaaggta caaagatttc ccatatatcc cctgtgctca cacatgcatg gcctccctat  28981 caccatcccc caccagagag gcacatttgt tacaatggat gaacgtatac actgaatata  29041 tcatattcac ccaaagtctg tagctcacat catggttcac tttggctgtt gtacattcct  29101 tggatctaga cacttttata atgacaggta cagtagtccc cctttatcct caggggctac  29161 ttccaagatc cccagtggat gcccgaaacc gcagagagtg ccaaacttga ctgccatcag  29221 tgggaatatg tttctattcg ccttccacca ccaccggttt aacgcctttt tcatcttagt  29281 gctgctgccg taactttggc agtttgagat gcgacagcaa aatgagtaca aatttctttc  29341 tccttcttca caatgtcatg gacagatgat tccttcttac catagatctt agcaacctcg  29401 gtgtgtgatt ttttttcttt cttgttaaat caactttcac cttttcactt aaaggaagca  29461 tttgacggct tctctttggc atatctgaat ttccagcatc acgactgtgc tttggggcca  29521 ttgtttgttt atttatttat ttattttatt tatttttttg agacagagtc tcgctctgtt  29581 gcccaggctg gagtgcagtg gcgtgatctc ggctcactgc aagctccgcc tcccaggttg  29641 acgccattct cctgcctcag cctcccgagt agctgggact acaggcgccc accaccaagc  29701 gcagctaatt tttttttttt tttgtatttt tagtagagat ggggtttcac tgtgttagcc  29761 agggtgatct cgatctcctg acctcgtgat cctcccgcct cagcctccca aagtgccggg  29821 attacaggca tgagtatttt atttatttat ttattttttc agacagagtc tcactctgtc  29881 ggccaggctg gagtgcagtg gcaccatctc gctcactgca acctccgcct cccaggttca  29941 agcaattctc tgcctcaaac tccggagtag ctagaattac aggtgcacac caccacgccc  30001 ggctRatttt tgtattttta gtagagacaa ggtttcacca tcttggccag gctggtctta  30061 aactcctgac ctcaggtgat ccacccacct cggcctccca aagtgctggg attataggcg  30121 tgagccaccg tgcttggcct tggggccatt attaagtaaa ataagagtca cttgaacaca  30181 aacactgtga tcctaacagt cgatttaatc accaagatgg ctataagtga ctaaggctgg  30241 cggggtgggg agcacagaca gcagggacac cctggacaag gggataattc gtgtaccaag  30301 caagacacag cggaaggcgc cagatttcat cgcactactc agaatggcat atcatttaaa  30361 actcatcgat tgtttatttc tgtaattttt ccatttgata tttggacagc agttgactaa  30421 gagtaactaa aacctggaaa gtgaaacagt ggataagggg gtgctcctgt acttctcaat  30481 gtgagacatt tgcccttcat gttaattctg ccccattagY tctacacaaa tgaatagcag  30541 gaaattgatt ttaaaccaca tggtggtaaa atgctttctt ttttctccct catttaacta  30601 aagaagggct gggccctagg tgatgtcttc cttagcatct aagcagctgg catcacccca  30661 ctgcttctgt gctccactct cccatgggac cctccctacc tttaatccct cctgtgctgg  30721 aggccgggtc ttccttgcta gtggtagctc tttccatatt tttaatccat ggtccggatc  30781 ctgctccact gcctttgctt tagagaaata aacatgaata ttgagtcact ggaaggaatg  30841 acacacgcat ccctccccca ccagttggag taacgctggc ccacctagtg tatctctggt  30901 ctaggtctcg aggacctgct gctcctccct cacctgtagt tgaagacctg cctcaggtca  30961 gtaggtgata cgcagtaagg aaatgtccaa aggacacttc ttgttggatt acacagcaaa  31021 catctaattg gctgcaaatc tttttttctt tttctttctt tttttttttg agacagagtc  31081 tcgctctgtt gcccaggctg gagtgcagtg gcgcaatctt ggctcactgc aagctctgcc  31141 acctgggttg acgccattct cctgcctcag cctcccaagt agctgggact acatgcgtgt  31201 gccaccacac ccagctaatt tttgtatttt tagtagagac gaggtttcac catgttggcc  31261 aggatggtct tgatctcttg accttgtgat ccgcccgcct tggcctccca aagtgctggg  31321 attacatggc tacaaatctt aaagggggaa gagatgagga ggaataatcc cctttgtctt  31381 ctcaaaaatg ttttcactgg ctcactacag ctccctcctt cctctttact gacccaaaat  31441 gccaactatt atagtaactc ttttgggtta gacgaatcca gtgaataaac acctactaag  31501 cattggaggt ccaagaggaa taagatatgc ctgagctaaa cctcatcacc cccggccttg  31561 cttgcagagt ggtttgctgg cctcatctga ttattcaatg agtgctttca tttgtttatt  31621 cactaaatat ttactgagca cctacaaaag tgcctggccc tggtagatga ggcctgaggg  31681 aagctaaaac taataagaca aactccatgc cctagaggaa ttcatggtct catagggaga  31741 aaatgtaaac acatcataaa attatagctt attaaatgct gcaatagagt actccgtaag  31801 agtgtggggg cagagaggag ggggagaaac agctgctttc tagagccctc accttttcca  31861 cttctctcat ctttctgggt tagggtctag cgggggttcc atgaggatca ggctaaatga  31921 gcttagaaaa actaagcaga ctactgtatc agaactggat cacagtagac aggcgttttc  31981 aacaaacata tattgagtat ccctgagtgc tttggacagg aagaggaatt atagacgaag  32041 attgtaagtc gcagtaatag atgaggcaag gagcaccagt gaggacttaa ccctgaaaga  32101 agtgtgagca catcttcctc ccagacaagg gggaataaag aaaggaagat gatcaggaga  32161 gttctaagtg gaactcagca gccagagggg aagcYggagg aggtaacatc agagggtgcg  32221 tttgcccact cagtaaagta ggaggcaggg cagccttgtg aaaataggaa tggaatagaa  32281 agctcaagaa aacagccaaa agcaagggca attaggggag gtttcgaact ggcagatcta  32341 cctcaactgg caatacagca agcatgcacc agaaaagata tcctagctag cagtggggtt  32401 gggaactgat ttaatatcct aggctagcag tggggttgSg aactgattta ttatttaatt  32461 ttttatatct attcatgtat ttatgcactt atttattttt gagataggct ctcgctctgt  32521 cacagaggct agagtgcagt gatgtgatct aggctcactg cagctttgac ctcctgggct  32581 caagtgatcc tcccacctca gcctcctgag tagctgggac tacaagcacg ccaccaccta  32641 ggctaatttt tgtatttttt tttgttagag acagggtttc accatgttgc ccaggctggt  32701 ctcaaactcc tgggctcaag cgatccatct gccttggcct cccaaagtgc tgagattaca  32761 ggcgtgagcc actgcgccca gccaatattt tttaaaaatt ggaaatccct tgtaataagc  32821 caagtgttgg gggaagaaaa gcaataaaag caatgacatg gactcaatat gaaacatcca  32881 aagcatttga catgccttta aaataaaaaa atcagtactc acctcgtgct catttcaaac  32941 tgtggatttc cttggtctaa aattttaaaa aacaaaaaac agcactctca aatttaagct  33001 aatttgaatt gattttacat agtgattttt aaatacacat atatgcataa agagaatact  33061 ataaaaatat ataaaggtgt gtaggtcttg atcatatttt cccaggaatt cagaaaacgc  33121 tgctacagtc cctgggctca cgtggtcctg gcatcctccc caacgtctct accccatcct  33181 gctgagtttc tggcatgcat tggttctggg tctgtccaag tttctactgt tagctccatt  33241 aatactgaat taaggaataa acagtgctca aatgctcatt ttttccatgt gagcccaaat  33301 tatgtgactt gatgagggaa aaaatcatga gtccctggga gagcgataag aatcacattc  33361 tttactaaag tgttgtccca ggtatgagaa taacaccaga tctcaacctc cagaggcccc  33421 ccactgcctc ccacagcata aaagccaaat tcctcggccc gatatttgag gccatctcaa  33481 tctttttcct ttcaccctat acctgactct cccaggctag gctcagatcg tcactgaatg  33541 tgctcacttc gaggcacctc tgtgattttc aaggttcctg ggcccacctt ttactaccga  33601 tgtgactgcc acatgttgcc cagttgctag gatgggaccg tggccttgat ttctgccggg  33661 actgaggctt tgccttgttt cccacccacc ctgctgcctg cccctgcact cttctggctg  33721 gggcctgatc tttccccgca gtctcccttc acctctagat cacaggcagt catgccacag  33781 ctgaggagct tgtcccaaac ctcagtgcct gcctccctcc tcggcttctt gtgctgctgt  33841 gtctcaccca tcagtgaYgc tcttctcttc cctgcccaag ccctagctga ctccataaca  33901 cctggataca atgtcctctt ctgtctggtt acctccgaga ggccctctct ctacatccct  33961 cattggctcc cagtgtcatt cctctcaccc atggccctaa ggtcactgta ttctttggcc  34021 agtgtacagg gttattatgc ttaacaatcc acaaaggttg aaaggtgttg taggatggtg  34081 taaaaatgaa tctgggtggt aatgtttata tgtcagagct ttgtaaagtg ctcggcaggc  34141 gtaaggtact gacagtcctg atattcctga tcttggaacc tgggacacca tcttctcaac  34201 attgcccgga taccctcaag ggtatccaga cagcctgagt ttgcattctg ttcctggttc  34261 agcccagggc cctggttccc gctcactcac catccactgt gggccctctc taaattctta  34321 aagcccttgc catttgcatc actcacagag acatttcatc agagcctact tggtgcacca  34381 ggctcaggag actcagttct gctgcggata agttatgtaa aattgaccct ctgcttgcac  34441 atctgtaaaa ggaaggggct ggcacaacac ctctgggcct ttcagttcag tagtgttttc  34501 tttttatcta aaccacgtgc tgggtcctgg ttttgcttct tatctagatt tttgcattcc  34561 tgtcacaacc tataaagcac agttcaggcc ttaaggaggg cttgagaaat ctctttccga  34621 attgtcaact gaataagtgt catatcttca taacaaactt gtcttttttt gcagggccag  34681 gaaggcagca ggggagtcag ttaaaatata aattttagat taagcttaat attgttaaga  34741 agtcaattct caccaaattg ttctagagat tttacataat cctaatcaaa atctcaataa  34801 ggttttttga gaagttggca agcagattct aaaatatata tagaagtata aaggacacag  34861 aataatcaaa acaactttaa aaaggaagaa caaaattata ggactctaac tacctcattt  34921 taagacttat tagaaagcag cagtaaccaa gatagtgagg gactgatgtc aaggtagaca  34981 aatagatcaa tggaaaagaa tcaggcatcc agaaatagat gcacttacat aaatcatcaa  35041 ttggagaaag aatagtgttt ttaacaactg gcaatggaaa aactagaaca tcagtatgca  35101 aaggactgaa ctttgatcca tacctcacac cacatacaaa tcaaaaacag aaacaaacaa  35161 acaaacaaac aaatactcaa aatggatcag agacctaaat gtaaaactat aaaacttctg  35221 gaagaaaaca cagggagaaa atctttatgg cttttagata acgatttctt aggcaggata  35281 ccgaaaacat gatacataca gtttttaaaa ttaaatatta taagaattaa agtcttttgc  35341 tcttcaaaag tcactcttaa gagaaaaaga tgccacacac tagaagaaaa tatttacaaa  35401 gcattaaaag gacatatatc taggatatac ataaaaactc tcaaaagtca ataataagaa  35461 aacaatgagc aaaagatttg aacagacact tcaccaaaga agagataaag atggcaaaga  35521 agcacataaa gagatgctca accattagtt actagggaaa agcaaattaa aacctaatga  35581 cataccacta tgcccctatt agaaatctga aaatttaaaa gactgacaat accaagtatt  35641 ggtgaggaca tggcacaagt ggaactctca tacattacta tgggaatgga agatactata  35701 atcactttgg aaactattta gaaatttcct aaaaaattaa acatacacct accatatggc  35761 ctaaccatta cactcttagg tatttaccca agagaaatga aaacacatgt ccacacaaag  35821 acttgtactt gcatgttcat agcagcatta ttcaaaatag ccccaaagca gaaacaaatc  35881 ggatgttcat taacaagtaa atggataaag aaaacggggt ctagccaaac aatgaaatac  35941 tactcagcaa caaccaaaat atgtactatt gtttacaaaa tccaaataga tgaatctcag  36001 aataattatg cagaggagag aagccagacc aaaaaaaaaa gtacatagtg tattatctct  36061 tatgaaattc tagaaaatgc aaactaacct atagtgacag aaaggagatt ggtggtcacc  36121 tggggtgggt gagggagggg caggagaaag ggaatgcaaa gcagtgtgaa caaacctttg  36181 gcggtgatag gcatgttcat tatccttact gtggtgatgc cttacagatg tatacagatg  36241 tcaaaactta tcagattgta cactttaaat atgtgtggtt tatcgtgtca ttatacctca  36301 gtaaaggagt tttaaaaatt gtagtaaaag gtctctacct agaaacctta ataagctaaa  36361 atgtatgtcc ccaggactaa ccctagctat tctatagttc tggggtggaa cctagaaatc  36421 tgcatttgta gcaagccctt caaataattt gaatgcaggt ggtccttaaa ccaccctttg  36481 agaaacactg acctagtgag taaggatttc taaacaagct tgtgactaga atgttgtttc  36541 tgcagggaac aagtgacttt ttctactaga gttgccatat cattctgtgt taagcctcta  36601 ggacactcct actcaattac catgacatat atttaacata atattaatag ttgtaaaaca  36661 aaagtaaacc atagtttttt tcccatctta tctgtataca aagtaaaatc aatgacataa  36721 tattgttact agttcctggc agttacctac agtttaaatc aagaacttat gttgcttggt  36781 ttttgtgaca gaaaactgat gtggtggctt tccagttaca ctgcttggtt taccttttcc  36841 cttatcttat gatgcacacc cagctttgca cgttcctgag cagctgatga tcatagaaga  36901 cctagaaaaa gtgtgagcca gttgcattcc tgctatctct aaagaaaaaa gttatctttt  36961 catttttatg tgatttttct tacccaaaat gcaaaccact gccagtataa atgatggaag  37021 atagactttc cacatgaaac tggcatttct tcaccctagt aacattacct gaggggaagg  37081 tcagaactca attgataaag ctcgctgtgg attttcccac caaatatccc tgcaaacaaa  37141 gtgaaaggat aaagcctgct taaagatgat ttgtaattgt tgaaaggagt acagtgtgtg  37201 acatcagtga aaatggtgga gaaaaaaccc gtccccaaat tctcttcttt gtgaaagcat  37261 ggaaaaaact ggccaaaaat ggttagaaaa tttattttca gaatatcgaa attttttttt  37321 cctttttttt ttttttatta tactttaagt tctagggtac gtgtgcacaa catgcaggtt  37381 tgttacatat gtatacatgt gccatgttgg tgtgctgcac ccattaactc atcatttgca  37441 ttatgtatat cccctaatgc tatctatccc ttccccctcc ccccacccca ccacaggccc  37501 tggtgtgtga tgttccctac cctgtgacca agtgttctca ttgttcagtt cccacctatg  37561 agtgagaaca cacggtgttt ggttttctgt ccttgcgata gtttgctcag aatgatggtt  37621 tccagcttca ccatgtccct acaaaggaca tgaactcatt gttttttatg gctgcatagt  37681 attccatggt gtatatgtgc cacattttct taatccagtc tatcattgat ggacatttgg  37741 gttggttcca agtctttgct attgtgaata gtgctgcaat aaacatacgt gtgcatgtat  37801 ctttatagca gcatgattta taatcctttg ggtatatacc cagtaatggg atggctgggt  37861 caaacgatat ttgtagttct agatctttga ggaatcgcca cactgtcttc cacaatggtt  37921 gaactaattt acagtcccac caacagtgta aaagtgttcc tatttctcca catcctctcc  37981 agcacctgtt gtttcctgac tttttaaaga ttgcctttct aactggtgtg agatgttatc  38041 tcattgtggt tttgatttgc atttctctga tggccagtga tgatgagcat tttttcatgt  38101 gtctgttggc tgcataaatg tcttcttttg agaagtgacc gttcatatct tttgcccact  38161 ttttgatggg gttgattttt ttcttgtaaa tttgtttaag ttctttgtag attctggata  38221 ttagcccttt gtcagatggg tagattgtaa acattttctc ccattctgta agttgcctgt  38281 tcactctgat ggtagtttct tttgctgcgc agaaactctt tagtttaatt agatcccact  38341 tgtcaatttt ggcttttgtt gccattgctt ttggtgtttt agtcatgaag tccttgccca  38401 tgcctatggc ctgaatggta ttgcctaggt tttcttctag ggtttttatg gttttaggtc  38461 taacatttaa gtctttaatc catcttgaat tcatttttgt ataaggtgta aggaagggat  38521 ccagtttcag ctttctacat atggctagcc agttttccca gcactattta ttaaataggg  38581 aatcctttcc ctatttcttg tttttgtcag gtttgtcaaa gatcagatgg ttgtagatgt  38641 atgatattat ttctgagggc tctgttctgt tccattggtc tatatctctg tttttggtac  38701 cagtaccatg ctgttttgat tactgtagcc ttgtagtata gtttgaagtc aggtagcgtg  38761 atgcctccag ctttgttctt ttggcttagg attatcttga caatgcaagc tcttttttgg  38821 ttccatatga actttaaagt agtttttttc caattctgtg aagaaagtca ttggtagctt  38881 gatggggatg gcattgaatc tataaattac cttgggcagt atggccattt tcacgatatt  38941 gattcttcct atccatgagc atggaatgtt cttccattgg tttgtgtcct cttttatttt  39001 gttgagcagt ggtttgtagt tctccttgaa gaggtccttc acatcccttg taagttggat  39061 tcctaggtat tttattctct ttgaagcaat tgtgaatggg agttcactca tgatttggct  39121 ctctgtttgt ctgttattgg tgtataggaa tgcttgtaat ttttgcacat tgattttgta  39181 tactgagact ttgctgaagt tgcttatcag cttaaggaga ttttgggctg agacgatggg  39241 gttttctaaa tatacaatca tgtcatctgc aatttgacaa tttgactttc tcttttccta  39301 attcaatacc ctttatttct ttctcctgcc tgattgccct ggccagaact tccaacacta  39361 tgttgagtag gagtggtgag agagggcatc cctgtcttct gccaggtttc aaagggaata  39421 cttccagttt ttgcccattc agtatgatat tggctgtggg tttgtcataa atagttctca  39481 ttattttgag atacgtccca tcaataccta gtttattgag agtttttagc atgaagggct  39541 gttgaatttt gttgaagacc tttactgcat ctattgagat aatcatgtgg tttttgtctt  39601 cggagaacac tggaaattaa atgatggctt gcagcaatct ggagagcatt tattcaagga  39661 aaatggctgt gtctcagtat gactaatgag ctttttaact tgccctattt ctatcctccc  39721 cttccttggt ggtagcctta gaaatgaaca gcctgcaatg atagtgaaaa tcagcagtct  39781 ggcagccatg ggaggggcag aacaggaatg ggggaactat ggagcctcat tcttagagaa  39841 ttatcattat ttgatctgtc cacgggtttc taggaatacc tgacctgcaa gtctgtcttt  39901 attaggcctg actcagaact tgcccaatgt gaaaagtctt ttccccaaag gcctttgtag  39961 aaaatgatta caggcaattg tttaacttct tggttgctcg aggtattggc taacagtggg  40021 gcaaacatgg gctaatcaga aggtttaaaa tgaaatgctc aggaataaga tgctcataga  40081 gggttgtaaa ggctccaaaa tatttatgag actctagaag accatgcaca catttcctgt  40141 gaacatgttc aggacagatc tgcgaaggcc ccacgatctt acctctggct gatcttgatg  40201 atctgcacaa acagaaagtg aaagctaggc tagaactgtc aagtgccagg ctgagtgtga  40261 aggtgtgccc taatgtgcac acagagcccc ttggcaaaga ctagaagact tactgctttc  40321 aggtgtttaa agaaatctct gtcatgtcat tagtatttag atcactaagc taactgaaca  40381 gaggcttcaa tggctgcaca taaatacaga cttcacagaa ttagtttaga aaagtcacta  40441 taacaaacaa caataaacag caacaccaac aaacagtaga ggtgggaagg tccaatttcc  40501 agagttgcta cattatgtta tttaaaatgt gcaattttaa cagaaaataa tgagacatgt  40561 aaagaaataa gaaaatgcag tccataccca gggaaaaaga aaaaccagtc aatacaaact  40621 gttcctaggc caggtgcagt ggctcatgcc tgtaatccca gcactttggg aggccaaggc  40681 aggcagatca ccagaggtca ggagtttgag accagcctga ccaacatgtt gaaaccccat  40741 ctctactaaa agaaaaatac aaatttagcc aggcatggtg gcgtgcacct gtaatcccag  40801 ctactcggga ggctgaggca gaagaattgc ttgaacccag gaggcggagg ttgcagtgag  40861 ctgagatcat gccattgcac tccagcctgg gctacagagc aagactctgt ctcaaaaaag  40921 aaaaaaaaag aaaaaagaaa aaactgttcc tgaggaagcc aagatactga ctttactaga  40981 ccaatacttt aactattttt acatgttcaa aaagttaaag gaaatcatat ataaagacta  41041 aaggaaagca caagaacaaa tcctcattaa atagaaaata taaagatttg ttttaaagga  41101 cgaaacagaa attctagatt tcaaaagtat aataattgaa tgagaaattc actagagtgg  41161 ctggctcaat agcagatctg agcaggcaga agaaagaatt agagaactca gaaataggtc  41221 aattgaatct atccagtctg aggaagagaa agaaagagga atgaaggaaa aatgaataga  41281 gcctcagaga ctgtgagata ccttcaagca cgctaatgac gcataatggc agtctcagaa  41341 ggagagaggg ataaatgggc taaaataata tttaaagaag caatggctga acatttccca  41401 aatctgatga aaacattaat ctataccttc aagaaagtct ataaactcca agaaatataa  41461 attcaaagag atcaagatcc acacctagag ataacataat caaactgtca acaaagacaa  41521 tgaaattatc ttgaaagcag caagcacata gaaggactct tcaataagat taacagctga  41581 tttctatcag aaatcacaga gttgagaagg caatgggatg acatattcaa agtgcttaaa  41641 gaaaaagagg gtcaacaagg aattctatac ctaaagccaa tttatcttca acaaagatgc  41701 caagaccatt caaagYgggg aaagaatagt cttttcaatc aatggttcta ggacaatgga  41761 tatccacatg taaaaaaagg aacttgggcc cctaattcac atcatataca aaaattaact  41821 cggaatgagt caaaggctta actgtaagag ttaaaactat aaactctttg caaagaacac  41881 tatcaagaga gtaaaaagac aatgcacagt atgggaaaaa atacttgcaa ataatatatc  41941 tgataaaagt ccagtatcca gaatatataa ataactctta caattcaacc ataaaatgac  42001 aagccaatta aaaaacatac aaatgagtta actgacattt ctacaaagaa gatataccaa  42061 tggccaatat gcatgttaaa agatgctcaa catcactagc catgagggaa atgtaaatca  42121 aaatcacaat gagatactag tacatgccca ctaggatggc aatgataata ataataataa  42181 taataatgtt attataatga acaatacaag tgttagtaag gaaatggaga aaattgaacc  42241 caactattat attgctggtg tgaatgtgaa atggtggtac tgttttgaaa gatgatttgg  42301 aagttcctca gaaagttaac aaaagttacc atatggtctg gcaattctac atatatacac  42361 caaagaaaac tggaaataaa gattcataca aaaatctgta caaaaatatc tacagcagct  42421 ttacttacag tatccaaaaa ggggaaaaaM cccaaatctt catcaactaa tgaatggata  42481 aacaaagatg gtatatccat acaatggact gttactcagc cataaaaagg tatgaagtac  42541 tgatagatac tacaacataa atgaaactta aaaaaatacg caaagtgaaa gaagctagac  42601 agaaagggcc atatatgtga tgtgtagaga aaacatccaa tagagacagg aaatacatta  42661 gtgattgcca ggggctgggt aaaggtgaga gatggagaat gattgctaat tggtacagaa  42721 tttttttgtt gggagagtta tgaaaatatt ttggaagtat aattagaatt aggctgttaa  42781 atactttaaa acaaaaaaca gctgggcgca gtggctcaca cctgtaatcc cagcactttg  42841 ggaggccgag gcaggcagat cataaggtca ggagatcaag accatcctgc ccaacatggt  42901 gacaccctgt ctctactaca aacacacaaa ttagccaggc gtgttggcgc gcctgtagtc  42961 ccagctattc gggaggctga ggcaggagaa tcacttgaac ctgggaggca gaggttgcag  43021 tgagcccaga tggtgccact gcactccagc ctgggcgaca agagcaaaaa ctccatctca  43081 aaacaaaaca aaacaaaaaa caaaagtaag aagtaaaaaa atagaattag actggggatg  43141 gttattgagc cttgtgaata atctaaaacc cactgaattg tatattttag acagtgaatt  43201 tgatggcatg ttaattatat cttgatgttt taaaaaaagt agtacagtgt ctgaaaggta  43261 gcattggcca tttgaatctc tgttaacatt ttcttgggtg gccatcagat ttttgattaa  43321 aggtgacctt ggcaattaag tggtgggggg aacacaacca aaagggacaa accaacacaa  43381 ccacacacaa tcacacaacc cctccacctc caccacagtg gaaatcaagc ataggtgctt  43441 ccagggactg tgaaaatgtg gccacagttg ttttcccagc aaaccatttt ctttcacaat  43501 atccccaatt tgcacatccc ccactgagct gaaatcagta taactcgaca gacRaaagtc  43561 agcaggggta acctaccttt gatattattt tgttgttcca tcaataattt acttatttct  43621 gaaaaccaac agtctctgat ttcttttgaa gctgcctgca atcacacata agaaaacact  43681 ataagactac catgagtatt aattgcagag tttttattct cccagtgacc gttagactaa  43741 tgagatagaa aacacttgtc aagtcatggg aacacaataa ggaaagagta gaggagaccc  43801 atggggaagg catgagcgtt acctgcagga tgtatttctc aagtccattt cgactggcaa  43861 tctcaaactt tctatggctc ccccttccaa gctggcgaat tgaaagtgtc atcagctatt  43921 ataaagaggg aagagaaatg ttctatacca tcgttttctg attttaaaat agtccctgtc  43981 aatcactcta atcctttcta tccttgacag ctctcttctt tcatcttcct ttcaagtttt  44041 tgctttcctc tcttaatgac tccaagacag cataaaacac aatctggtac agtgtcttct  44101 gtctcatctg tttcagaagg taacacagac tgttaacttt ctactgtatc tttatgcagg  44161 aggcgctgca gccctcaacc tttcagtgaa gagatcattt ctccttcaac Rtgtatttta  44221 caactttcta cgtaatagta actgaataag gatctattca gaaacataaa taacaaggcc  44281 aggtatggtg gctcatgcct ctgtaatcag tcctgtaatt ccagcacttt gggaggccta  44341 ggtgggcgga tcacctgagg tcaggagttc gagaccagcc tgaccaacat ggtgaaaccc  44401 tgtctctact aaaaatacaa aattagctgg gcatgctggt gcgtgcctgt aatcccagct  44461 acctgggagg ctgaggcaga attgcttgaa cccgggaggt ggaggttgca gttagccaag  44521 atcatgccat tgcactccag cctgggcaac aagagcaaaa ctctgtcgaa agaaaagaaa  44581 gaaagaaaga caagaaagaa agacagaaag aaagaaagga aggaaggaag gaaggaagga  44641 aggaaggaag gaaggaagga aggaaggaag gaagggaggg agggagggag gaaggaaggg  44701 agggaggaag gaaggaagga aggggagaaa gaaaacaaag gctactatga acaaattaac  44761 agctttaatc acaaSagcat aaatatatat gggtgtggct cctgtatgtg ggtgtgtgtg  44821 tttcatagcc tgagagctaa ctggctatga aacagtttct aaacaggtga gaaaacaatg  44881 cttggtgtgg gcctgggtgc cttagacccc aaggtcagga caatgcctgc acttagacac  44941 aaagatacac cgttgagaag gRcaagtcta agatgaaatc cagcccatgt tctcatcact  45001 aagtctttca actctctttt acctctttgc ctggaagaaa gagggtactt ttactaatta  45061 gtttgtccag actggctgcc atatagtctt ttttctctag tacaaaggca ctcctatgcc  45121 agccctggcc cataagctct gaccctggag tcagacagac ccggctatgg attctagccc  45181 tgtggtgtgg tatttggcaa gttaacttca cctctctaca ctgcagcttc ctcatctgta  45241 aaataaggaa aacgatctct ctcttttctg catagggcga ctcccagatg caataggact  45301 ccctttacct tcatggYctt cttgaagctg taatgaggag ataatccctg gtccccaggc  45361 tccattcgta tcttacagaa cactattccc ctttcaaata ggtagatttg cctctggctg  45421 ggtttaaatc gaatcaaatc cttcatttta taacgatcct tgtgaattgt ccagacgctg  45481 aaagggccgt gcagcaacag cttgcctagt tttccaatat cgtccttttg gaaacacaca  45541 tacaggaaaa gaagctgtaa aattacttga cagagaaaga agcctttgtg gcctgttttc  45601 aaattttata aagcatgctt ctttgaggtt tacaattcta aaagtgctat tcaggctggt  45661 ctgtcactaa atcaaaacga caattttttg agcttcactg agcgtggtgc cRtttagtta  45721 tataaggaaa tcaattcagc tatgaaaggt aatgctatac ataaagaatg ttagaaatgc  45781 ttgagctaga gatacgggga ctgtgatatt tttgactcaa ttttaactag cagaattcaa  45841 tttcctctgt gctatgtttg atacatgaca gtctccttca aagggacaag aaattaacat  45901 ttcaattatg tccagtagag atagaatttt ggctgcattg Yggacccatg agcatctgtg  45961 tgttccatct gcatcctaac ttctgcttca ggtatatgag gacctgagaa aatgaagctt  46021 gttctgaaaa gagaaagttt actgtgatct aatcttcaca acattgtaat ggaaatagta  46081 actgcattca actctgagtt tttcagagtg agtcaacagt gccaagataa taccaaatga  46141 cagattttta agaagtctgt ttgactctag gcataaactg tgatgcccct gtctccacca  46201 gcaaggctgc ctttgtcata tgtctggttg aaagtccaaa tgagatgacc ctgccttctc  46261 tgagctctct tcaaaagaca gtggaccaag atgagctggg agcgaggcat tttatctggt  46321 atttttcaaa acttcgattg cttaaagcag ataagaattc atttccctga ataaaaacac  46381 caaaaggaac aaattatttg aaggggacca agacaaagaa atctccctaa gtgacctcta  46441 gctgagatca atccctatga tttctgagtt aatactggaa atatgtactt cattcataat  46501 tgattgggca tttttataaK ttattgttta tatgttgggc aaactctagg tatctttttt  46561 aaaaaggaat tgactttact tctgaaacat taaaagaaaa acttcaagag caataaactc  46621 tagtcatttc tatggtgtct atggcgttat ttcctatgtt aattttctat tttcttcttt  46681 ggaaaattta catatgtaga ctgtggtgaa taaaaccctc aaactcccag ccacgaggcc  46741 ttctttccta aaagccaatg ctcctgttct gtgttaacag ttcaccacat tccaaaacag  46801 aagggtggat aggtccatga ggaccctgga gaattttttc actctatctg catccatcaa  46861 ggacccactc aaatgtaatc actacagacc ttagtgactg ctttttcctt taaatccatt  46921 caattcataa tcaatatcga cataaatagt gcaggaaata tgttttagta aaggcaactg  46981 gtagaagtag atacttaata tttgccacat aaacaaatgt tatcagcaca ggttgagcat  47041 ccataattta aaaaatctga aatcctccaa aatctggaac tttctgagaa ccagtatgac  47101 actggaggtg aaaaattcca tacctgacca catgtgatgg gtggcagtca aaacacagtc  47161 aaaattttgc ttcaYgcaca aaattatttt ttatttttta tttttttgag acggagtctt  47221 actctgttgc caaggctgca gtagagtggc acgaccttgg ctcaccgcaa cctctgtctc  47281 ctgagttcaa gggactctcc tgcctcatcc tcccgagtag cttggattac aggcgtgcat  47341 taccatgccc ggctaatttt tgtattttta gtagagccag ggtttcagca tgttggccag  47401 gctggtctcg aactcctgac ctctggtgat ctgcccacct cagcctccca aagtgctggg  47461 attacaggca taagccactg ctcccggcct tcatgcacta aattatttaa aatatgtaaa  47521 attaccttca gtctatgtgt ataaggtgtc atgaaacata aatgaatttc gtgtttacac  47581 ttgggttcca tctgcaacat aactcagtac gtatgcaaat actccaatcc tcccctaaaa  47641 aaaaaaaaac tccaaaacac ttttggcctc aagcattttg gctaagggat attcaaactt  47701 gtagtttcta tcttacaatg tattcactct ctacctcatt ttaaacattc gacgcatctt  47761 acagaaatat atacagtata gacagataga aaataaccga gggaattaag gtgaagaaaa  47821 aataaagaag ttgggagctt ggcgcctgcc tgtagtcgca actacttggg aggctgaggc  47881 aggaggatca cttgagtcca ggagtttgag gccagcctgg gcaacatagg gagatcacca  47941 tccctaaata taaagacaat aaatcagatg aactgttcca gaaggtgtag aagataaaag  48001 aaaagatgaa gaggttatat gtaaaatgca tataataaag ccatKtaaac ttgatgtaaa  48061 cttggctctg actttctaat agcaatacag agggaaacat ggtgagtggt acgatttatt  48121 atgtctgaag acatacacaa accggttgtt caggaaaagc acagctattc ctactaagac  48181 ataagaaaca tttctcccag tgatatgttg tcactgagca acatcctcaa ccacgcccta  48241 tgatagaaag aggaacatgt tttaggtaaa gctgcttgta agaacacatt tggtcagtgt  48301 ggaggctcag ctgtcaaagt aattctagaa gaggctagga tgatagggga agggggaata  48361 agtcgaggtg tacactctct aatgacttgt gaagatgggg tgacgtgagt taaggcatcc  48421 acaaggctat tgtagaggaa acagccccaa ccagaaatgc tgaagaggca gcctcaggca  48481 gcaaggtttg tgctttatta cctctgaact ttcctcatca ccccatccac agccatgctg  48541 ctgcaccttc ataggcaagt aatcagagga tgtgaatcca gagaggtacc agcagctgac  48601 catagggccc ccagcataaa aacaatgtgc cagggKtggg ggttggcaat gtgatctctc  48661 cctgaactga agaaaacagg aggaagtgRt gaccagcagg agaYggggct gtgagatgct  48721 ttaggggaaa gcaggcagtg gaataaacga gaaggaaaca gaactatgaa gaaggtgagt  48781 cattagaaat tatgaggaag aggaagaaac tgacggaagg gaaaaaaaca gtcagtaggt  48841 ggtaagagSR ggggcgagca ggcatgtggg tgcacttgac atatacaggc gggtcacaca  48901 ggtaagaacc ccggctccct ccccaggccc aggccccaga ggtgctgtgg atcctgggtt  48961 tgctcagttc ccatctccgc gaacttggct ttgccatcgt gacttcttga gaagctgcag  49021 gtcagctttc cagtaagccc attattactg gaagtgactc ggggggagtc gctgggtctt  49081 tcaagtctgg cagaaacata tgggcacata tcctctgggt catgttgcac aaatgttctc  49141 aggaccacac atatgacggt attgcccatc tccacgccca aggtccgatc tccagcgggt  49201 gagcacatgc cctacacggt gctgaccaag ggcaggcaca cacactgttc actgtgctgt  49261 gtggagctgc tccgcctcca acaggaagct gcagggacat tccctgagca ggagcaagaa  49321 tgaccttttc cgtcacgtgt accttttctt tccaactaga ctgtagttga tctcaaggtc  49381 ggagaccacg ttgcacaacc tctttcagat acgtgttcac cagtatttat ttatttattg  49441 gcattcccta gtatttattt atttattggc atttagtggt gcttgggagt aggggacagg  49501 aaacaaaata ctcatcactg tgagggaata ttgatttatc caaggagttc attcagcgac  49561 gcccctgtac tgggttgaat agtgttcccc caaaaactca tgtctatgag accctcaaaa  49621 tatgatctta ttcggaaaga gggtctttgc agatacaatt ggttaagatg gggttgccct  49681 gggttaggat gggtccaaat ccaatgactg gtatccttat aaaaaaagaa aacaaagaaa  49741 tggagacgca cgctgaggga acagccatgc aaaggcagag gcagaaattg gagtggcaca  49801 gctgtcagcc aaggaactcc aagaattgcc ggcagtcacc agaagctcag acgaggtaag  49861 gaaggattct tcctgagagc cctcagaggg aacgtgggcc tgctctcacc ttgcctttgg  49921 acgtctagcc Ktcaaaactg tgagggaata aatttctgtt gttttaagtc acgtactttg  49981 tggttccttg cgacatcagc cctaggaaac gaatatactt ccaactgttt tccaccagta  50041 acaaagaagt gtggccatgt cacacggtgt gtggaagaac ctgatggttg tgaactcaga  50101 gatggaacat agcggctacc tcaaatgagc gagcgataag ttagatggcc tcctctgtac  50161 aagaagtgat agcaaaatca attagtaaat gctaccactt tcacacctgg gcattctgca  50221 ggtaagagcc ccgatacaag gatttcaaat gtgactacat tggccaaatg aagagtctgg  50281 ctgagagagt gaatctaggg aagctggcct cagcaagagc ttcttttggg tgcccgtgtt  50341 tccattccag gtagtaagag ttaatatgac tgtcacaaac ttactgcaca aagaagcagg  50401 aaacatctgt ataaatgcgt ccacacagag cctgagtcca gaaatgaaaa gcagaagctg  50461 tgggagcctg tgattacaat agtcaggggc aaagaggtat cttcctttcc agcttgagca  50521 ggtccccata taacgtacca ctagggctgt ttattcggca tctgttgggc gccaggcaca  50581 gtgcttggtg ccttacccat catttaattc tcacttactc ccggcaaccg cctgtaatat  50641 aggtattgtg ggggggtggg gggggtgggg gggtggggaa tgaagaagat actttgcctg  50701 ggtcactagg attatttact tagccctatc tctgcagcta acactgtggt aaggaaagag  50761 gaaactgaaa tagaaataaa atcctggccc tagctcacaa tgaacttaca atacaagtac  50821 atcctttagt ggtttatata aatttgttcc aaaaaggaag actgtttcat tcaatcttta  50881 caagtcaact ttggaactta tccctggcaa gtcagaattt aaatgcatct ctaatatgca  50941 ttggatttga aaaaaaaaaa aacttttgtt ttttttttgc aaatagatag aagtaaatta  51001 taactcagtg ttccacaaaa caaacttgtt tggaaaagaa ttttaaactt ctgtgattaa  51061 attagaccca ggactaattg agtacacaga aaaacaagaa aatataaata tcagaaggga  51121 tttttttcta gttgctttct agttgtttaa ttgtgataat ttaattatca cattcaattt  51181 ctttccttta taaaaccagg ttaatattaa atttcaagca gagaaactaa gaggttaaac  51241 tggactcagc catgcatata gctgtaattt attacaggtg atcataaaca cctgtaataa  51301 aactctacaa aatggagaca aagaatctca gaatcatttg agctgagtta gaggcagttg  51361 tcagtcaatt ctaatcagaa acagataaag agtggaatag caccctgata gggaatgaac  51421 aagaaatagt ccaaggtgac agccatgtaa tcttgagtga gttaactctt ctttccatgg  51481 atcttctttt gaatcagaag gttgaaaatN ccaatatcta ctcctNcata attcaacagg  51541 attatacaaa actgtcagga gaaattagct gatgtattag atgtaaaagt aagttaagtc  51601 ttagtagaaa aggtaccaaa aagcatctgg cagaggcaca attctgagga cacatggtca  51661 aaaaaggtcc tttgcttttt gcgtgtaaaa tctccaaatt ctgtaagctg gttcatccta  51721 tttgcatgca aactggctgg gaatgaaatt agggcagRat gttatttgct catgttttaa  51781 ccattctttt ctcacagcct cctcctccta tgttatttac actgtttcct gccctgtgta  51841 ttgtttccag gacattcatt agattcaggg aaatgaaatt taatagggat gtctaatacg  51901 taattcaaga tttaaaaagg aacagaaaga tgccctggat tgacctaaca aattgttccc  51961 ttgactttcc tcagScgaag aaagaagaat tattaccaag aaaatgatcc ttatacacac  52021 cctaacccta ctctgcagtt tatgcatatc ctcttgaatc atgcatcagt tgtcatcaga  52081 gacaccttgg agtcccaggg gacagtaaca gcatcatggc tgggatcaca gcaaacccca  52141 tggggctaag tttctgagat ccaggcccaa gaaataccag cgtgggaagt aaaaataact  52201 catttttcgg accttaataa ctttcatggt ttgatttatg acaaggaaag aaaagtcatt  52261 tcctctacca agacctcaat acatggtatg ttcaaaacca aataaaaatg aaaagaaaaa  52321 aaaagagaag gaaaaagtat aatataatca caagtgacaa aaacgcagaa gagggggtca  52381 cagagatgtg atgtaagaaa aacttggccg ggcacgatgg ctctcacctg taatcccagc  52441 actttgggag gcggagatgg gtggatcacg aggtcaggag ttcgagacca gcctgaccaa  52501 catggagaag ccccgtctct actaaaaata caaaaaaaaa aaaaaattag caaggtgtgg  52561 tggcaagtgc ctgtagtccc agctactcag gaggctggga caggagaatc gcttgaacct  52621 ggcgggcaga ggttgcagtg agctgagatc atgccattac actccagcct gggcgacaga  52681 gcaagactct gtctcaaaaa caaacaaaca aaaagaagaa gaacccaacc tgccattgct  52741 ggtttgtgct ttattacctc tgctcataga agccatgagc catggagcgt gggtggcctt  52801 tttaagctgg aaaattccag gagacagatg ctagactcca aaaaggaaag gaaacacctt  52861 gattttagcc catgagactg tgttgaactt ctgaccttgg aacttcaaaa taataataat  52921 aatgaatttg tgctgctcta agccaccaag cttatggtaa tttattatga cagtgaaata  52981 aaacaaacac atctgaataa aaatctagag tacttcaaag taaaaaacaa aacaagtcga  53041 atattaacta tgctccagtc tcaaagagat catacagtta taggggatgt tttgcctgga  53101 atgcctggga ccagcctgct ccggattaac actgacgctg gagctcaact ccccagcatt  53161 ggcctctaac tgcatctctt gggttttctt tagactattt ggattaatgc atgtacacta  53221 tatacttttg gggcctcttc tgggatctgt gatataaaat tggggcaaga atataggacc  53281 tgtttccaaa atacttttct tagagaaact ctgaaacaag aagacaaatc ttttactatt  53341 ttaatagtgc catgagttag aaaataaatt gctcctccag gacttttcct ctgcccatat  53401 atgttctggc ccttgacttt ctggtttgtc cagacagtgg gtcagtccca cattcagaac  53461 cctgaggaat aaatYcaaga ttcctgaaga tgccaaggga cagcttggaa tcttgtagtg  53521 cgggtttact gagcaattta ggcgactgtc ctcagagtcc aattccttta ccatagacga  53581 tctctctgac tttgccagag catttcctca ctgtgtagta ttgcacctgc cttttgtgtg  53641 agatgctcta acactactat ggaacatacc ggacattcag tcactgctgc taggtccaca  53701 gccaactcac aggacttgat caaatcctct atcactgcca aagcttgttg tagttcagtt  53761 gagaatgctg aatctttggt tctctttggc ccatcctgtg gaaaacaaat gccttgttgg  53821 aaatcaaaag tataccatct gcaaagtgaa gaaggaaaac aggacagaga tgaggagcca  53881 gggatccagc caaccctgac tgagaagggt ggatattgat ctgtctccct gctcccaaag  53941 catggtggct aaagatgctt actgatttag gtatacggca tagagacaag caaaatcatc  54001 tacctctgtg cattgtacta gtgacgaatg aagttcataa atcatggaaa ttatattcct  54061 gctacataag aaacatatag aattcagcta tatagctgaa ttctatatgt ttcttatgtg  54121 tatatatatt atataaatat ttatatagta tattaaatat ttatatatta atatataatt  54181 atatgtttat atattaatta tatatattaa atatttatat attatatatt atataaaata  54241 taagtatata tattatatat atatatatat atatatatat attttttttt tttttttttt  54301 tttttttttt tttttttgag agcgagagag agtttcactc tgtcacccag gctggagtgc  54361 agaggcacga tctcggctcg ctgcaacctc tgcctcctgg attcaagcaa ttctcctgtc  54421 tcagcctccc gagtagctgg gattacaggt gtgcgccacc atgcctggct aatttttgta  54481 tttttagtag agacggagtt tcgccatgtt gaccaggctg ttctcaaact cctaacctca  54541 ggtgatccac ccaccttggc ctcctaaagt gctgggatta caggcgtaag ccactgtgcc  54601 tggccagaat ttagcttttt taaagcatgg aaaaacctag ctccttttaa ataggcttct  54661 ctctctttta tacccctatt ccctggggct tactgaaaaa aaaaatacca tatggccggg  54721 cgggtggctc atgcctgtaa tcccagcact ttgggaggct gagacaggca gagcacttga  54781 ggtcaggagt ttgagcccag cctggctaac atggtgaaac cccgtctcta ctaaaaatac  54841 aaaaattagc ttagtgtggt ggtgggcgcc tgtaatccca gctacttggg aggttgaggc  54901 aggagaacag cttgaaccca ggaggcggag gttgcagtga gcccagattg tgtcattgca  54961 ctccagcctg ggtaacaaga gtgaaacccc gtctcaaaaa aaaaaaaaaa aaaaaaaaaa  55021 aaagtagctg ggtgtggtgg tgtgtgcctg taatcccagc tactcaggag gctgaggcag  55081 gaggattgct tgaacacggg aggtggagtt tgcagtgagc caagattgcg ccactgcact  55141 ccagcctggg caacaaagtg agaccttgtc tcacaaaaaa agaaaaaaga aaaagaaaaa  55201 gaatacaatc tgagtatcat attcctatat ttaatgtgga agccttattc ctgtgggaaa  55281 aattatactt aaaatcatcc cagaggagga catttgtaaa ctcctataga gacaaagaac  55321 tccatagagg ctgctaagtg gaaatttact aatgatttac atgtaaaagc tataacatca  55381 tatttccaca ctgaatctcc cccaactctg tccttccttc ctccacttgt ccctggccct  55441 ccccacattg caccaccatt aaagatgcca aagagataag ccagcgctct gcacctcccg  55501 aacataaaga ctcagcattc agcRgaaaag cagtaactaa ttaaagagac caatgttcca  55561 attacaacca cagtgacatt taggatgtga ttggggtgat tgtttcagct ctaaaggctt  55621 ttgcatgggc ttgaggtatt ttatctccct gctacctaca tgctgtattt atctgttacc  55681 tggtaaatac acataaaaaa attttggttt attcaatgta tttttttaac atctcaagtt  55741 ctgcagtgaa gaacagctag cccctttgct gctccgcatc tggccctgac tctttttgtc  55801 ctctacagca caatgtagtc acagggttta atattttctt aatctatgcg gaagcactgg  55861 gtatttgcat ctttggatga gagacatgag tgacagggct gatgaatgga atagatcgtg  55921 ctgctgcctc tgaaaaacgt atcatggatt cgtagcttct cattcataat aaagaagctt  55981 cgcttcctgg taaaagaaaa cagactctca cctgaattta ttttaacaac atcaataagg  56041 gattaagagt tcttggcagg gcgcagtggc tcacgcctgt aatcccagca ctttgggagg  56101 ccgagggggg gagtggatca cgaggtcacg agatcgagac cagcctgacc aacgtggtga  56161 aaccccatct ctactaaaaa caaaaattag ccgggcgtgg tggcgggtgc ctgtaatccc  56221 agctactcag gaggctgagg caggataata gcttgaaccc gggaggcgga gttgcagtga  56281 gccgaaactg cgccattgca ctccagtctg ggtgacagag cgagactctg tctcaaaaaa  56341 ataaaaaaat aagaaataat ttttttttaa agttcttata ccaggcatct tcctccttac  56401 caaacactga atgctcagtt tcctttcaga tcctcacctc ttcccatccc ccttccttcc  56461 gctaaccctc cttcacaaaa cacgaggtgg ctagggtttt gaactgtata atgtagggtt  56521 cagaaaaatc ctttaaaaca ttattaaact cctctaacta gggctagctc ctcttgctcc  56581 aggctgtaaa aatatgacct gtgtcctgag ctgcttctgt tttcaacagg tccttgtcat  56641 ccattctgag cagaaagggc atgcaaatga ctgccccata agacatgtct caagtgtttc  56701 ttgctaaaac cagaatattc tataggaaag ggaagagaaa ccgcactgct ataMcacaga  56761 tttcttccta acctggtgtg gtcatggtca ccatttattc taagggaact ttggcagact  56821 cttagagttc acacacacgc acacacacac acagaggaga acaaggcata acatatagaa  56881 attagatata ctaagaggta caaagaagaa aacataatcc taccattcag gaaagccaca  56941 gcagacattt tatcatatct atttacttcc agtcttttat gtatgcattt tacgtatttg  57001 cttaattttg ttcccattaa aatttttttg gctgggtgca gtggctcacg cctgtaatac  57061 cagcactttg ggaggccgag gcaggtggat catctgaggt caggagttca agaccagcct  57121 ggccaacatg gcgaaacccc gtctctacta aaaataaaaa aattagctgt gcatggtggc  57181 gggcacctgt aatcccagct acttgggagg ctgaggcagg agaatcgctt gaacccggga  57241 ggcagaggtt gcagtgagcc aagattgcac cattgcactc cagcctgtgc aatggagtga  57301 gactttgtct aaaaaaaaaa aaattatgtg gaaggaagaa aatatattac cacttccatt  57361 tgggctgcaa atccctactt taaaattgcc tttaattttt ttgttttttt tcttgactgt  57421 gagttatagg atacatacta ttttagaaaa tctagataat aaagaccacc aaRtaaaatt  57481 gctataaatc ctccgaMcca tagacaaaca ccattaatag ttagttatat aaaatgtaat  57541 atttaatatt gagtacctta gccgagcctc ctagttcacc tgggtctatc tccatatctt  57601 caggagactc gaRatccagc agaccctgca ttaacccaaa agtcttgttg aaaattaaaa  57661 tgtacattgc catcaaaatg tagattgtca acaaaatcta ctgtgcaaac tatctgccac  57721 caatgctaag ctgactgaga acgtgtgtaa atgtgtaaga gggaaagaat aaatgatgta  57781 tttggcacag ggtcctttcc acaaacctag aatcatattg tacacactat tttgtagact  57841 actttttctc attcaaccat acctcatgaa tattttccca tgatattatg tttttctaaa  57901 atatgggctt tgattgtggt agagtgttat atcttacaga taaaccattt ttctcattca  57961 gcaatatctc atgaatattt tcccatgata ttatgttttt ctaaaatatg ggctttgatt  58021 gtRgtagagt gttatatctt agagataaac catttttctc attcaacaat atctcatgaa  58081 tattttccca tgatattata tttttttaaa acatgggctt tggttgtggt agagtgttat  58141 atcttataga taaaccataa tcgaattata gtccccctta tcattaaaca tttaggttca  58201 acaatatttt ttactataat aatgataata acttttgtgc atttttctta aagctaaatt  58261 cctagagtgg aattgtaaat gttaagagtt ttgaggcata tttccaagtt cttcagagaa  58321 ggactgtctt cctgccagct gtataggaaa gcctgtctca ccagccccta gcctacaagg  58381 ggtagtacca tttaatcaaa aatctttacc aattgaatga aaagagaaat cctacctcac  58441 tgttgtttta atttgacttc cttagataac tagaggtgta gaactttttt aaaaaaacgt  58501 ttgctactag tgtttattct tttgcaaatt gctatgtcct ttgcgcatct ttttattata  58561 atttccaaga gcacatgatt cattaagcat actgatcatc tgttacatat tttgttcaca  58621 ttttccccaa tgtttcatga gtctgtcttt aatggtataa gctatcactc atcaccctct  58681 ccccaagatc ccatgatcct ttctaaagca tgaggcaatc agtccaacat tcatgctctt  58741 tcaagccagc acatgtgtgc gagatgcaaa ataagctctg cccctgggga tagagaaggt  58801 cctagatagg attacaaggt Ygttcctttc tttgcaggca cgtaatggcc tgagctggtt  58861 tcacaggcac cagcaagctt agctgtgggg acactgctct ggtctgcctt gggtagctcc  58921 cacggctcct cacagacccc ccgcaaaata ttttgtagta attctatctc ttgtttcttc  58981 agtgaaaggc agctaacaaa accttctaag tccttttaac ctgtattatc tcatttaatg  59041 ctcccaacaa gcataccagg aaagtactat tattatctta tttctatagg tgagaaatct  59101 gaggctgaga gaggttaagt aacttgcctg agttcacgca gcctggaagg gtcagagcct  59161 ggattcagac ccaggttgtt aaactctcga gtctgtgttt ccaatgacga tgccccacag  59221 ccctctgcgt ctggtaccga acctagctcc tatgtaaatg tcacctctgt gggaaagctg  59281 gaacctaggc tgagggagga aggaccatca ctttcgtcct gctcatgcct cactgggccc  59341 agaggttgga cttctacaat aggaatgaca gtgacaatgg gacatgcaga ggagcatgag  59401 gccctgagtg agtgctgggt caaacatgct ctgaggtgct gctcgctgaa agaagcagat  59461 cattatcctc atttctatgg tggtgtgaca gtctcaatgt ctaagataac agctacacat  59521 catgcagagc ttcctgggct gtgccaagcc ctccgcatgt gctaatcatc tgcttcataa  59581 cgatcttatg gaggaggttc tagtaccctc attttacata gaggaaaact aaggcccaag  59641 aggKcaaggt ctcataactg acaaatggca gagtacaccg aggctgaaac tgctaatgga  59701 aagcttgaaa ggagtaactt tgaaaaaatg tttttaaaat tatttttaca gatgaggtct  59761 tgctatgttg cccaggctag tcttgaactc ctggactgaa gggatgcctc ctgcctccac  59821 ctcctgagta gctgctggga ctacaggtgt gtgttatcat gtctggcctg aaaggggtaa  59881 ctttttctga ggggagactg ttgggcaatg agcttaacct tcctgagcct cagtttcttc  59941 acagtccaga gcatggctgg tatgtctgat tgtggagatt aaataagaaa tactgtaatc  60001 ccagcacttt gggaggctga ggtgggcaga tcacgaggtc aggagttcaa gaccagcctg  60061 gccaacatgg tgaaaccccg tctctactaa aaatacaaaa attagccagg catggcggcg  60121 cgtgcctgta atctcagcta cttgggaggc tgaggcagga gaattgcttg aacctgggag  60181 gcagaggttg cagtgaactg agatcgcgcc attgcactcc agcctaggca gagtctcact  60241 ctgtctcaaa aaaaaagaaa gaaagaaaga aaaaagaaat acacgtgggg aagtacccag  60301 taaacagtag gtgcttgttt ttgtttttgt ttttgttttt tattaagaaa tagtggccac  60361 tgggcaagag ctacctaaat gccttataga ggttaattca acagacaact tatcgtggtg  60421 gcccaaacct ttctctcaag ctcacaccta gaaaaccagc catgataggc cctcatatcc  60481 tgtttggagt gtctgagaat gcttgcctgc agatgtgcta gtgtcaatga cttatcacta  60541 aggaggggta aaggtcagag agagagagag aaaaccactt aggggttcag ggttcagata  60601 tagcaaagtt cattctgtta ggctgctctc agggtaagat cctaaatcta agaatggagg  60661 tcagtgctgc aaaaggatag aagacttcag ttttgtcttc tcccttccac aagttaaaat  60721 gcccataaaa cagtaacttt ggggaaaatc acactctcgc tcccaaagag ctctcttccc  60781 ctaagccaga ctccttagtg attcatgccc tgagctagac attggactag tgaggccctc  60841 aggtgccctc caactctatg attctttggt cttactccac attgaagagg gctgatttag  60901 agttggagga aagaaaaagc ctgcaggata gacgtatctt cagccttaga agaccaaggc  60961 acagctctac atgtgtaacg acatgggcac cacagtgcag gcgtctggtt tcccaccaag  61021 gaaaagcact gtctcattga ttttaccttc aacagcatct ggtattttat aagtctctgg  61081 cttggtcctt tgagatactt aaaaagaggg agattgtgat ccagttggcg ctggcatacc  61141 tttaaaagcc aaatagaaac aacataaagc aaaacaaaca acacacacag aataaaagtg  61201 gcagcattta ggtacaaaac acaaaattgc ttttcctgga caacaggggc agctgtgtcg  61261 agcagctcca taaggctgtg gggttgttct ttcccctagg aacagtgtgt gaaataagag  61321 aaagaggaaa gaggacggct cggctaatat ttttacaggc atctagggaa gcagaaccat  61381 ttatttcttt tagaaagcaa gttcactaga agacagtggg gcatttcaag tccttctgaa  61441 aaagaggcca cctatcttca agttgggggc tgccagagtc tattcaggcc ttttggagcc  61501 tgaaggatac agacggtttg tagacatcat tccagggagc ccaaagtgtg acatttttta  61561 cttagtttca tttttaaaac ccatcttaga agaagacact ggttttctca ccagatatag  61621 gtgctcctca caggcctatc ccattacaac ttttcatgtg taatttcaaa gtatgggaca  61681 ctttaaataa acataaatcg caatgccaag ttcctctccc tagtcccact tttgatttct  61741 ttaacaactt gatgcataga aacattttcc aggaagaagt ttgtcttctc aacattaccc  61801 caaagtaggc gcagtcttga cactcttgcc agattgccct agctcggggc agattcttat  61861 ggtatttaaa atatatctga agatcttctt tctaggtggg aaaaaattag aataaattaa  61921 tcaggcagca tttttcagta agtggacagg atgatacagt ttattcattc attcaacaag  61981 ccagagtatt ctaatgtgta aggaattggg ggtgtacagt aagtaatctt atgttgggat  62041 gtgtgtgtga tggagagata aacagaaggc tttaaacaac cataactacc agacatagat  62101 aggcacagag tgctctggga gcatctacaa gggctattgg taaatacata agtagactca  62161 caaacaggaa ttggttttta tctttatcat tcttttcctg ccctttgtcc aggacatcga  62221 catctcccca caacaggtct gcaatgagta gtaaaactga cagaaaagag gctggtgttt  62281 gatggtcccc ctctcttctc tctccaccct ctccttgtgg tcatcgtgac accaaccctg  62341 caaagaagcc caagaaacaa ccaaaagatg gaaacatgcc agaggggaga tgtggtagat  62401 cgggcaacca atacggagct cagtcatttg ggcaaccctc aaagtgacag gctccgcctc  62461 aaacatcact ttcaacctgc agcacaagac tctgagacag gaaatgttat gtgactgttt  62521 cggagctaga gagcaaggag gactcaacgt gctgttcaac ctgtattctc atggtatcaa  62581 agacgctgag aggtgggcac atatctgcca atctcccgga gagaaagcac actccaagcc  62641 tctggcgtca gcttgccagc ttcactcgct ctatcaggga gcctggccca cagcggctgt  62701 gctggccaca gatcccatgc cattctggtg gagaagccca tgtagccacc tccttgcaga  62761 agtctgtgtc cctgtcctcc tgctcagccc aactctggat ttccttcatg tcaacacaga  62821 tgattccttc cctctcagct tcaatgatgc ctgttctact cccaaataca ttgctatagt  62881 ggatgacttc catttctgta gctctttctt tctcccaata ggttgtaagt ggtcatttca  62941 gcataggaac tacagcaacc gtcctttgct attccctcca ggaaaaaaat ggcacacaca  63001 tttaagacag atggcccttg aaaaatgttc agactggtta aggagctaga ataataactc  63061 agagcttaac aatatagtat tgacctaaat tgctcttcta aaggctaaca gtccaaaatt  63121 cctcagagaa aggatgtcca ctaaccagca acatacaaaa gtgcccaatt ctcctaatct  63181 ccttatagaa tgaatcagga ggctataaaa ttgctaccag aaactcatat tcacacaaga  63241 atacatactt gcttacggat taagactgcg gggctctggt ggcggatggt ccaggttcag  63301 ttcttggctt tgccaaatac aaatcatatg gcctaaagta agttacctaa ccttcctaga  63361 cttcagtttc ctcatctgtt aagtgggccc ataacagtag tcaactcctg tgactgttgt  63421 gagattaaat gagatgatgt atagaaacct ctccacaaat gcccaatgga tagagtactc  63481 aaaaaatggt ggctattgtc aaagcacttt ttactttatg gaatcctctg catacattat  63541 tttgtttccc acaacaactg tacatattgg tactattagc ctgttgtgtg gaggaggaaa  63601 gtgagattcc aaagtttaag ttatttactg gaggtcatgc agatagcaag tggcttgacc  63661 aacatgataa atcgggtttc ctgacttacc agttactccc acatatcagt gtgagcaccc  63721 attccagtgc tctttccatt ttaccatgtg agatctcaga gtacttggaa cccacagtac  63781 actatggaca aatattattc tatatagaac atggtctctg gaggagaatt gtttcagaag  63841 atacaggcta aagaggctga ttaaaacact tggtcagaaa actatttcaa aattcattat  63901 cttggaaggc cgaggtgggc ggatcacgag gtcaggagat ggagaccatc ctggccaaca  63961 tgatgaaacc tcgtctctac taaaaataca aaaattagcc gggcgtggtg gcacatgcct  64021 gtaatcccag ctacgaggga gggtgaggca ggagaatcgc ttgaatcagg gagtcagagg  64081 ttgcagtgag ccgagatcac gccacagcac tccagcctgg tgacagagcg agactccgtc  64141 tcaagaaaaa aaaaaaaagt actacttgta ttttgtctct aacatcataa atcacatagg  64201 gctaagtcag tgttttctgg ctgggttaat gatttacaat gttcctccca acatggcggg  64261 gcgctatcaa aaaactttgt aaaactttac aatttaggag aggaattcag aaaaggtatt  64321 tagttaataa ttctctgaca atttcctcat atttgaacaa tttgataata tccactttcc  64381 cacaggaact ttgcccattt ctcattgagc aatttaaata atctttgcag taatgacata  64441 cataaccata aaatacttct aatctctgaa actagatttc acattagtcg tttgttaagt  64501 gcaaaaattc aaaatagtat ataaagcact aataatgtaa tagttcaaaa aaattaatga  64561 atcagaatga gtataaaata gttttcagtg tacattgaat tctggtgctt tgacacaaag  64621 cctatgatga gttgacatgg aaagccatat ggggccacca aaaggctctg agcatggaag  64681 attccttctc tccattaatg tcaaatatct tgaaacctgg aggctttgtt tgcttccagt  64741 tatcataagt attgctctca gcctcagtca taatatgagt tgcttacaac tttaagttgt  64801 ttgatctgtt gccttgggtt cacaaaaata aaaataagaa agagaatgag aaggactagt  64861 tgagaaagag aggagaaatg agaactattt agaaggcaca gggggtcagg atcatctcca  64921 tggtcgtgtc ctagaaagta cctggtgggg attgatgttg ggtggaacat ttctctgtgg  64981 cagctcccac tggcccatat agcctatttg ttgcctaaaa cagggtggag aaacagggaa  65041 agagggaaga agtctgggag gtgggaagaa ggggtagttg gaggtataat gttgtaggcc  65101 aagatgttca taaattgagt actaagccta aaggaggctg tagatttaga attttcgaat  65161 catcctctat gttgtaaaaa atagaatgga aagaaaatgt atttcctgag gtttttagtt  65221 taacaccagt gcttctcctc ttgagagcat tagtacagct ttattgtggg actctaacct  65281 cgctgtatga ttcttgtttt aaaacgtagt tgagtcattc agaagaggat cctctgcttt  65341 ggggaccatg gatggccagc cttccatttc atggggcttt gcaggaagcc aggcttgcgg  65401 ttactcccac atatcagtgt gagctcagtc aaattggact cattctcacc ttggataaag  65461 tcagataaag ttgggaggcc aactcatgtt ttccattcaa agggcagata caagatatac  65521 tgtcttgaca aattgcaagt gtacagtaca gtattattcc atgatagtcg ccatgctgtg  65581 cgttacatct ccaggactta ctcatctcat aactgcaagt ttgtaccctg tgaccaacat  65641 ctccctactt cctctagact ccagcccctg gcaaccaccc ttctacttca atgagctcaa  65701 ctgtttttgg attatatata tatacatata cacacacaca cacacacaat ggtaatgaat  65761 gtattcatta acttgattgc agtaatcatt tcacagggtg taagtatatc aaaccatcat  65821 gttgtatagt ttgaatatat gcaattttta tttaccaatt atacttcagt aaagcttgga  65881 gtcggaggga agcagatatg tttttagacc attgatgttt ccatttgtcc ttgccactta  65941 acgcagaaat gtaagggctg actttaacat gattatttac tgggagtaca gacttactct  66001 cttgagaaag caatgtgcca gaagttcagg gttctcagca cacttttcca actcttttag  66061 aaaagtccta gaaaaataaa agtatacaag taataatgtt ttgattttga catgtaggta  66121 attaattttt taagtacaat cacataaggt ttcaagaggt gcctagaaaa atgttctcca  66181 ggccttcttg ctcttatctt ctacacattg attcagRaaa aaaagctgta ttctgaggta  66241 ttactacaat taccctgaaa tcactcacaa ctttaagttg ttcgatctgt tgccttaggt  66301 tcacaaaaag aaaaataaga aagagaatga gaagaactag ttgRgaaaga gaggagaaat  66361 gagaactact cagaaggcac agggggtcag gatcatctcc aattatcaga acacagctgt  66421 tcgggtaatt ctaagcacta gtcatagata gactggatac attcactgat gctctctctt  66481 acatgaaagt aacgttcata agaactgctg ctgctgctgt tgctattact aatatcattg  66541 acagcttact ataagccagg cataagctaa gtgctccgca aatagtatct catttaatcc  66601 tcaaccaaag atagggtttt ataaatataa aacctggggc tcaaagaggc tacataactt  66661 ctctggggaa atcaacagaa atgggtttcc atttcagttt tgcgtctgac caacggacga  66721 ggaatggggt taggaagcaa ctgtggttca attcaccaag cagcttttct ccctctgtga  66781 attaggtgtg caatcttggg gtcatcatag cgaataaaga gagttaacaa gttaccattc  66841 ccatatgtgt aacatgaaag ggtttgaaga tgatttccaa agtcccttat tcctgcaatt  66901 ctctagaact caatatccaa gcagccccga gttaaaagta caacttgttt gagcagtcag  66961 cattttctaa caccctagtt cccagcatcc ccttatgata gtagtatgga gatgtgtatc  67021 ttggtcatct ttgtattccc aatgctgagc acagtgcctg gcacagaaca ggtgctcaaa  67081 aaatgctgat gcatgaataa ataaacaaag gaacactcaa ctgcatacaa catggatgtc  67141 tgacactggg tccctgttcc tttgatgtct cttccctttc tctccaggga tcctgtgggg  67201 cttctcccct cctttcactc caacccaccc caatccaatc cactgactcc aaaccacttg  67261 cttaaacctg aaaacatcta agtgttttca tctctcaatt ccatcagtct catttccacc  67321 cttctSttac tctccattct ttctgataac atgaattatt atatacctgt tgtgaaattc  67381 gtaaagttct ctaatattcc caaagagaaa gtccttgtta ttctgaagaa catctggaat  67441 tagatgcttt agccaaataa aatccattgg agtgatatat ccctgcagag gtgcaaacaa  67501 ggtaggtgtt acaaacagga acataccaga gatcatctga attagctact gcaaaaccca  67561 aaactttagt gaaatgattg aagaatatag cacacttgtt tcttgaaagc tatcgtattg  67621 agtactttct tttgatatca ttttcctcat gtgcccactt tcagaaggag atggcttaat  67681 ggcaatgatc ataaaatggc atttttgtgg gaagaacaaa tatatttaaa aatgtttatg  67741 ttacacaggt ttaatctaaa agaagggaaa atgtacccac tgttttatga actaatacaa  67801 ttactattat tattatgatt attattaata ttgagacgaa gttttgctct tcttcccagg  67861 ctggagtgca atggcgtgat ctcggctcac tgcaacctcc gcctcctggg ttcaagtgat  67921 tcttccgttt cagactccca agtagctgga attacaggca cacaccacca cacctggcta  67981 attttcgtat ttttagtaga gacagggttt caccatgttg gccaggctgg tctcaaactt  68041 ctgacctcag gcgatccacc cgcctcggcc tcccaaaatg ctgggattac aggcgtgagc  68101 cactgcatcc ggcccaataa ttatttttta atatctgaat aatgtttact cagcatatat  68161 atcatagttt actaaacatt ccctttttat ggacatttgg gttgcttcta atattttatg  68221 gtaaaaatgt gattataaat aatcgcgtac atataatttt ttccttaaga ttattccctt  68281 aggaaaatag ggaaaacatg catttttact cttaagaaaa agactacttt aaaaaaacaa  68341 gaaataatct tcccttatta aagtatcagg aacacttata atatcctttg ttggcaagaa  68401 gatggtaaac ctatttgggc tttgctggaa tcagtgtgaa ctggcttaat tctttggaaa  68461 ctatttggct acatggatca agaaaccaca gggatgttct tattcctact ggagacactt  68521 gtaatattct aagacctgaa accttggtgg gaattctgga gacttctggc aactattttg  68581 agtctctttg tacaacaaaa catctgcagt ttgaatatat gcttgatacc ccactcccca  68641 aatctactta catcaattat gcttttaatc tcttttatgt aaatctcttc agtctcaagc  68701 aagtcacgta taatgcgcct gaatcacagc agcaggtggg agggtgaaag agagagagac  68761 agggagagga gaatgttctt ttagaattct gttagaataa tgctcatcaa tacagttccc  68821 ttttagtggc tcacctactt agtttctggt cagttcattc tgttctattg aacacccaag  68881 gtcagcatct cacaaacgca cactgtgatc acactggtat caagaagaaa cagcaaggtt  68941 agagaactca aaatccttta ggcagccagt gaatgacctg tcctctgggt gggccatata  69001 gtgtgggggt caagaaggga gatgttggag tcagaaatac caggtctgga tccttagtca  69061 aggaaggtct gcctgacccc acccccaccc cctatattgt atacattgta taagtggttt  69121 cctagatttt ctctccagct ttctgctcac atcaagcttt cttttccttt taagagaaag  69181 ggtcttgccc tgttgcccag gaatgagtgc agtggcatga tcatggctca ctgtagcctt  69241 gaactcccgg gctccagcaa ccctcctgcc tcagcctccc aagtagctag ggctacagat  69301 atgcaccatc acacccagct aacgcctttt tttttttttg gtagagatga ggtcttgcta  69361 tgttacccag gctggtctca aactgctact ttcaagcaat cctcctgcct ttgcctccca  69421 aactgctggg attataggtg tgagacactg tgccaggcca gatccagatc tttgacccat  69481 atggatgtat tttgttgtac atggggttag atgtatgcta gctccttcct ctggcattgg  69541 atgtttcact gtgatttcct aaggcaaaag atatgacttc tgtctgcttc tgtcaaggaa  69601 tgcaggtgga agatgtggtg gacagaattt aagacggtcc ccaagacttc tggccccact  69661 gcacatacgc ctcttccaat caaacactaa tttaggggat gctgtggaag gattttgctg  69721 ttcatttgat aggtaatcca gttgggcctg tcctaatcat atgaactcag atctatMtgg  69781 gtcaagtggt cagagactgg aagcataaaa aagattcaac acaaaggaga ttcccctttg  69841 ctgacttttg agatggaggg ggccagatgg aatggaatgt gggcagccgt aggaactgag  69901 agtggctccc agctgacagg cagcaaggaa gggaaatcag tcctatagtt gcaaggaacc  69961 gagtcctgcc acaaccacgc gaacttagaa gaggatccag agctccacat gaaaacacag  70021 ccagccaaca ccttgacttt agccttgtgg gactcctgtc ccagggaaac tgtaggtctc  70081 ctaagtttgt ggtcagtttt tatatagcaa tagaaaacca gtagaaagag taaggccagt  70141 ccccacatct atcccagaca gactttcttt ctctcctcag gctacgtggc catatttatt  70201 atttctttta ggagtccaag tagatgagtg tttatacatg tgtccttgtg tagaatatat  70261 atatttattc ttgttaacta ttgatggatt atctattaaa tggcagcctc tcctagaaaa  70321 tgatttgttt ttctctcata aaaatggatg tgacaaatga tccaatagaa aaaaatggga  70381 aaggacacaa aataggagat tcacacaaat attcaaatgg atatgagact cgtggaaaaa  70441 atactcagta catacattca acgaaggcat attttaaaag agcaacaatt ttcatctatt  70501 agaatattaa acatttcaaa cctcatagaa agactggtaa atgagggtgt ggaaaaaccc  70561 tcacacaata ttggtaaaat gtgtaaattt gtgcaagctc tttggaaggt aatttagaaa  70621 catctcaccc aaatgtaaaa cacgtgctcc ttggttcagt aatttcactt ctaggaatgt  70681 tcattgttta taattaggac aatataactt caacaacaac cagatataaa ctggttactt  70741 aaatgctcat caaaaaatta tggtacatgg gccgggcatg gtggttcacc cctgtaatcc  70801 cagcactctg ggaggctgag gcaggtggat cacttgaggc caggaaatcg agaccagcct  70861 ggccaacatg gtgaaaccct gtgtctacta aaaatacaaa aaattagatg ggtgtggtag  70921 cgcatgcttg taatcccagc tacttgggag ggtgacgcag gataattact cgaacccagg  70981 aggtggagat tgcagtgagc cgagatcaca ccattgcact ccagcctggg caacaagagt  71041 gaaactccat ctcaaaaaaa aaatatatat atacatacat atagatatag atagatatat  71101 gtgtgtgtgt gtgtatatat atatacatat atatggtaca tacatataat gatgaaatac  71161 catgcaattg ttaaaaagaa tgaggctgac taaaacactg atatgacaga ggccaggtat  71221 ttttagtgaa aaaacaaaac aagacgtaga caagtaagca tagtatgact atttgtgtaa  71281 aaaatgaatg tgtatgtaga aatatacgta aaactgtata tgctcagaaa atttatgcaa  71341 agaaacttaa cacattgtta aaagtgattg cttttggaga gtggtactga aagctaaagt  71401 atgggaaagg agaatttcta catcttactc tatacccttc tacaatcttt tttaaagtga  71461 aacatttact gcttttataa tggaaaaata gggtttacat aatattttaa aatgaatgtt  71521 actggaggta atgatatgta aaccctattt gatagatata aataaataat acaattcaca  71581 tattttacag ctcattattc tttcttataa tcatttttgt gtctatttat aaatacttgt  71641 ataagacaag actgataaac aagatgtacc acagaggatg taatttccgg aagtctaaaa  71701 ctcccaaaaa gttacttaaa catcatactt tactaagtca ctgagaaaga ggttcctatg  71761 ccttttataa atcacccgga aacaacaacg gtttctatgt cagaaggaag tacaggcctc  71821 aataccaata tgtttctacg agccctggca tcaagtggga ccacagcaac agttaagcat  71881 tcatggaaaa cctgagtaca atcagcctct tatgccttca tgaagcactg ggcttaagca  71941 acttggcaag ctggcttcag gatgcattga caattaaagg aagtcagaag gcagaaataa  72001 ccaggaggtg gacagaagtc aaggttatta tccaaaaatg atctatgaac ttaagagact  72061 cttaggagct tttaggactc taatacagga aaatattcac atctctgaag gaaaagaaat  72121 cccttggtca tattctggat tacagtttgg aaaaaaagtc tagaaatctc atccaactgc  72181 ttcattttac ctgttggagt gcaagagata aaatgccaag gataggaact ccactgaaga  72241 atgtaggcaa atcaatattc attaaataca ttcaaattaa tatatgactt aaaaagagcc  72301 ctcacatcag tgttaggcca ttgcttttcc ttgcttctct tgttttcctt tcaaatacct  72361 ttataaaata ggagaaaatg tatgactctt tttttttttt tttttttttt attatactct  72421 aagttttagg gtacatgtgc acattgtgca ggttagttac atatgtatac atgtgccatg  72481 ctggtgcgct gcacccacta atgtgtcatc tagcattagg tatatctccc aatgctatcc  72541 ctcccctctc ccccgacccc accacagtcc ccagagtgtg atattcccct tcctgtgtcc  72601 atgtgatctc attgttcaat tcccacctat gagtgagaat atgcggtgtt tggttttttg  72661 ttcttgcgat agtttactga gaatgatggt ttccaatttc atccatgtcc ctacaaagga  72721 tatgaactca tcatttttta tggctgcata gtattccatg gtgtatatgt gccacatttt  72781 cttaatccag tctatcattg ttggacattt gggttggttc caagtctttg ctattgtgaa  72841 tagtgccaca ataaacatac gtgtgcatgt gtctttatag cagcatgatt tataatcctt  72901 tgggtatata cccagtaatg ggatggctgg gtcaaatggt atttctagtt ctagatccct  72961 gaggaatcgc cacactgact tccacaatgg ttgaactagt ttacagtccc accaacagtg  73021 taaaagtgtt cctatttctc cgcatcctct ccagcacctg ttgtttcctg actttttaat  73081 gattgccatt ctaactggtg tgagatgata tctcataatg gttttgattt gcatttctct  73141 gatggccagt gatgatgagc atttcttcat gtgttttttg gctgcataaa tgtcttcttt  73201 tgagaagtgt ctgttcatgt ccttcgccca ctttttgatg gggttgtttg tttttttctt  73261 gtaaatttgt ttgagttcat tgtagattct ggatattagc cctttgtcag atgagtaggt  73321 tgcgaaaatt ttctcccatg ttgtaagttg cctgttcact ctgatggtag tttcttctgc  73381 tgtgcagaag ctctttagtt taattagatc ccatttgtca attttgtctt ttgttgccat  73441 tgcttttggt gttttggaca tgaagtcctt gcccacgcct atgtcctgaa tggtaatgcc  73501 taggttttct tctagggttt ttatggtttt aggtttaacg tttaaatctt taatccatct  73561 tgaattgatt tttgtataag gtgtaaggaa gggatccagt ttcagctttc tacatatggc  73621 tagccagttt tcccagcacc atttattaaa tagggaatcc tttccccatt gcttgttttt  73681 ctcaggtttg tcaaagatca gatagttgta gatatgcggc attatttctg agggctctgt  73741 tctgttccat tgatctatat ctctgttttg gtaccagtac catgctgttt tggttactgt  73801 agccttgtag tatagtttga agtcaggtag tgtgatgcct ccagctttgt tcttttggct  73861 taggattgac ttggcgatgc gggctctttt ttggttccat atgaacttta aagtagtttt  73921 ttccaattct gtgaagaaag tcattggtag cttgatgggg atggcattga atctgtaaat  73981 taccttgggc agtatggcca ttttcacgat attgattctt cctacccatg agcatggaat  74041 gttcttccat ttgtttgtgt cctcttttat ttccttgagc agtggtttgt agttctcctt  74101 gaagaggtcc ttcacatccc ttgtaagttg gattcctagg tattttattc tctttgaagc  74161 aattgtgaat gggagttcac ccatgatttg gctctctgtt tgtctgttgt tggtgtataa  74221 gaatgcttgt gatttttgta cattgatttt gtatcctgag actttgctga agttgcttat  74281 cagcttaagg agattttggg ctgagacgat ggggttttct agataaacaa tcatgtcgtc  74341 tgcaaacagg gacaatttga cttcctcttt tcctaattga atacctttta tttccttctc  74401 ctgcctgatt gccctggcca gaacttccaa cactatgttg aataggagcg gtgagagagg  74461 gcatccctgt cttgtgccag ttttcaaagg gaatgcttcc agtttttgcc cattcagtat  74521 gatattggct gtgggtttgt catagatagc tcttattatt ttgaaatacg tcccatcaat  74581 acctaattta ttgagagttt ttagcatgaa gggttgttga attttgtcaa aggctttttc  74641 tgcatctatt gagataatca tgtggttttt gtctttggct ctgtttatat gctggattac  74701 atttattgat ttgcgtatat tgaaccagcc ttgcatccca gggatgaagc ccacttgatc  74761 atggtggata agctttttga tgtgctgctg gattcggttt gccagtattt tattgaggat  74821 ttttgcatca atgttcatca aggatattgg tctaaaattc tcttttttgg ttgtgtctct  74881 gcccggcttt ggtatcagaa tgatgctggc ctcataaaat gagttaggga ggattccctc  74941 tttttctatt gattggaata gtttcagaag gaatggtacc agttcctcct tgtacctctg  75001 gtagaattcg gctgtgaatc catctggtcc tggactcttt ttggttggta aactattgat  75061 tattgccaca atttcagagc ctgttattgg tctattcaga gattcaactt cttcctggtt  75121 tagtcttggg agagtgtatg tgtcgaggaa tgtatccatt tcttctagat tttctagttt  75181 atttgcgtag aggtgtttgt agtattctct gatggtagtt tgtatttctg tgggatcggt  75241 ggtgatatcc cctttatcat tttttattgt gtctatttga ttcttctctc tttttttctt  75301 tattagtctt gctagcggtc tatcaatttt gttgatcctt tcgaaaaacc agctcctgga  75361 ttcattgatt ttttgaaggg ttttttgtgt ctctatttcc ttcagttctg ctctgatttt  75421 agttatttct tgccttctgc tagcttttga atgtgtttgc tcttgctttt ctagttcttt  75481 taattgtgat gttagggtgt caattttgga tctttcctgc tttctcttgt aggcatttag  75541 tgctataaat ttccctctac acactgcttt gaatgcgtcc cagagattct ggtatgtggt  75601 gtctttgttc tcgttggttt caaagaacat ctttatttct gccttcattt cgttatgtac  75661 ccagtagtca ttcaggagca ggttgttcag tttccatgta gttgagcggc tttgagtgag  75721 attcttaatc ctgagttcta gtttgattgc actgtggtct gagagatagt ttgttataat  75781 ttctgttctt ttacatttgc tgaggagagc tttacttcca actatgtggt caattttgga  75841 attccctgct ttattattct aaagcaagct tgtccaacct gcggcctgtg gcccaacaca  75901 aatttgtaaa ctttcttaaa acgttatgag attttttttg cgattttttt tttttttttt  75961 tttagctcac cagctatcgt tagtgttaat gtattttttg ttttgttttg ttttgagacg  76021 gagtcttgct ttgttgccag gctggaatgc agtggtgcag tctcggctca ctgccacctc  76081 tgcctcccag gttcaagcaa ttcccctgcc tcagactccc gagtagctgg gactgcaggc  76141 gtgcgccacc atgcccagct aactttttgt atttttgtag agatggggtt ttaccatgtt  76201 ggccaggatg gtcttgatct cctgacctcg tgatccaccc tccttggtct cccaaagtgc  76261 tgggattaca ggcgtgagcc acctcgcctg gccagtgtta atgtatttta tgtgtggccc  76321 aagacaattc ttcttcttcc agcgtggccc aggaaagcca aaagattgga cacccctgtt  76381 ccaaagcatg taattttatt cacagaaaag actctcggcc gggcgcggtg gctcacgcct  76441 gtaatcccag cactttggga ggccgaggca ggcggatcac gaggtcagga gatgtagacc  76501 atcctggcta acacggtgaa accccgtctc tactaaaaat acaaaaaatt agccaggcgt  76561 ggtggtgggt gcctgtagtc ccagctactc gggaggctga gacaggagaa tggcgtggac  76621 cccggaggtg gagcttgcag cgagccgaga tcacaccact gcactccagt ctgggcaaca  76681 gagcgatatc cgtctcaaaa aaaaaaaaaa aaaaaaaaag actctcaagg atttaccatc  76741 taaaatcatc aagtgtatat catgcagatg aacaacagaa aacactggga gtattcaagt  76801 aattaaaaat aacctttcag caccaacagc aatttctgtc ctgatggcaa tctactcaga  76861 gctagaggac tgcacttagg ataccaaagg gagcttagga tgcaagagca gcctgcacta  76921 atgtactctg cgttagttta catccgggca ctttgcttta cgtttcaaaa cagacgccgc  76981 ccttagctca ttaaagggga aatggaggta taatgtgttt taaagggatg tttcttctct  77041 gaagccatat ttcagggtag catgaaaaca gagctttgga tatctggttc cattctacat  77101 gacacctcat gcttgtttca aatgacacca caaccaaggg gcaggaatga aaggaatatg  77161 ctaaaaaaaa aaaatgcaat cctttttagc aagaaagatc attaaggatt tcctgataag  77221 tttcttcttt gttcatgtgc cctcctctgc tccccctttg aactactgcc cctgtcaccc  77281 ccccttcccc gcctccccgc cgtttctcag ttctccaata gggatccagt ctgccagagg  77341 tctatttttg ctcattattg tcaacacaat ttgccatcaa gagttttcac ttactcctgc  77401 tatcctatca tcacattatg catgtcagaa agcattacag atttttactt ttcaagaacc  77461 cgaactcaga tttccaggct tctgtccttc ctgtttcatg ctgttgactc tcccacatct  77521 ttgtcttcct cctagacctt tggtcactct tgagcccaca ttgccaatgg tctatatcat  77581 tttcacctgg atgcctccca gacacctcag tcacatcaag ttgaaataga actaattaga  77641 cagaagtagc atgtgtttca gtggaactga catttttcta gggtccctaa aacacaggtg  77701 ttgtttcaaa ctcccccctc caacatccct tatattctct ctgtcaccca ggctccttat  77761 atcatctctc aacatgcttc ctgggtgact actgactctc cacttctggt gacaccccac  77821 agagcaagct ttcaattcct gtcatttaaa cactttagtg cactcataga atctcatcct  77881 tctgagattt ctcttccacc agtctatttt gcagaccacc aggctacgcc cttccaaaag  77941 ctcctttgag atggagtttc actcttgttg cccaggctgg agtgcaatgg catgatctcg  78001 gctcattgca actgccacct ctcaggttca aatggttctc gtgcctcagc ctccccagta  78061 gctggggtta aagttgcctg ccaccatacc cagctaaatt ttgtattagg ggttttacca  78121 tgttggccag gctggtctcg aactcctgac ctcaggtgat ctgccaccta ggcctcccaa  78181 agtgctggga ttacaggtgt gagaaaccac gcctagtccc ccaaagctcc atttttatcc  78241 tgtcttcatt ctctcttcaa gaacttgccc acagctgcca gagttccttc taagagttta  78301 agactctctg ttcatgtggt aagaatcttc ttaaggagct tgctttaaag gtaaaaccag  78361 atttacacat cagaatgggg agaggtgggt gggagctaag atggaatctg taattttcac  78421 aagtgccctg gatgattcta ctgcagatgg tctcctattc aactaaatag cactcatctg  78481 acccactcct tttagacccc acttctgtta agccagtgtt atcgctattt tccactttct  78541 ttcaagttct aatcatcctt aaaactccaa cagcttcagc tgaaatcagt accacactct  78601 catccttgtt tcccacccaa aaactattgt actaattgac cagacatgta ttgatgccct  78661 actatgagcc tggctccatg ataggcactt gggatagaga aatgtaccat aatcctgacc  78721 ttaataaatt tagagtctaa tggaagagat aaaaaaaatc aatgattcca atgagtgtga  78781 taaggtaagg ctagtcagaa aagtcaggtg cagaggaggg gcaactaatt aaaccagggg  78841 ttgggggaga cactgccaac tctgtgttga gtgaagttta agcagaggtt tttctttaag  78901 taaaaaatca gtttttttaa ataaaaagtt ttttaaaaag taagggggaa tagaagaagg  78961 aggtttgtcc ctaaggggaa cagtgtggag gaggaaagga ggtgaaagtg tatgctattc  79021 tgtgtactgg caaattcttt ggtgggtgat atggtttggc tctgtgtccc cacccaaatc  79081 tcaccttgaa ttgtaataat ccccacatgt catgggaggg acccagtgga aagtaattga  79141 atcatgaggg caggtttttc ctgtgctgtt ctcatgatag tgaataagtc tcaggagatc  79201 tgatgatttt ataaagggcg gttcccttgc acatgctctc ttgcctgcca ccatgtaaga  79261 catgcttttg ctcctccttt gccttccacc atgattttga ggcctcccca gctatgtgga  79321 actgtgagtc cattaaacct ctttccttta taaattaccc agtctcagct atgtctttat  79381 taggagcatg agaatagact aatacagtaa attggtactg gtagagtggg gtgctgcagt  79441 atctgaaaat gtggaagcaa ctttggaact gggtaacagg cagaggttga aacagtttgg  79501 agggctcaga agacaggaag atgtggtaaa gtttggaact tcctagagac ttgttgaatg  79561 gctttgacca aaatgctgat agtgatatgg gcaataaagt ccaggctgag gtggtctcag  79621 atagagatga ggaacttgtt gggaattgga gcaaaggaga ctcttgctat gttttagcaa  79681 agagcctggc agcattttgc ccctgcccta gagatctgtg gaaatttgaa cttgagagag  79741 atgattcagg gcacctggca gaagacactt ctaagcagca aagcattcaa gatgtcactt  79801 gagtgctgtt aaaagcattc agttttatgt attcacaaag atatggttcg gaattggaac  79861 ttatgctcaa aagggaagaa gagcataaaa gttcagaaaa ttggcagcct gatgatgtga  79921 tagaaaagaa aaacccattt tctgaggaga aattcaagcc tgctgcagac atttgcataa  79981 gtaagatgga gccaaatgtt aatcaccaag acaaagggga aaatgtctgc agggcatgtc  80041 aaggaccttt gtggcaaccc ctcccatcac aggctcaggc ctaggaggaa aaagtggttt  80101 tgtgggccca gcccggggac ccctgctcta tgcagtctag ggacttgttg ccctgcatgc  80161 cagttgctcc agccatggct gaaaggggcc aaggtacagc tcaggccatt ggttcagaga  80221 tgcaagcctc aagccttggt ggcttacacg tagtgttggg cctgtgggtt cacagaagtc  80281 aagaattgag gtggatgtac agaaatgcct ggatgtccag gcagaagttt gctgcagggg  80341 tggggccctc atggagaatc tctgctaggg gagtatggaa gggaaatgtg gggctggagt  80401 ccccacacag agtgcccact gggacactgc ttggtggagc tgtgagaaga gggacaccat  80461 cctccagact ccagaatggt ggatccacca acagtttgca ccatgtgctt ggaaaagctg  80521 cagataccca acaccagccc atcaaagcaa ccacaagggg ggctgtaccc tgcaaagcca  80581 caggggcaga gttgcccaag gctgtggtgg gaacccacct cttgaatctc tatgagacat  80641 ggagtcaaag gagattattt tggaacttta aggtttgact gctttattgg atttcagact  80701 cgcataaggc tgatagcccc tttgttttgg acaatttctc ccattttgaa caggtatttt  80761 tacccaatgt ctacacccac attttatctg ggaaatagct aacttgcttt tgattttaca  80821 ggcacatagg tggaagggac ttgccttgtt tcagatgaga ctttgaactg tggacttttg  80881 agttaatgct gaaataagtc tttgggggac tgttgggaag gcatgatttg tcttgaaatg  80941 tgaggacatg agatttggga ggggccgggg tgaaatgata tggtttgtct ctgtgtcccc  81001 agcttcatca ggatcctccc acacatcccc attctaagtt gcagggtccc attcttttcc  81061 aatcaatgcc ctcattttaa cagtagacac ctggtgaggc tgtgcatgca cctttcattg  81121 caggtcagcc gctcccatga taagagcttg tatctgattt tccacaattt cagctctttc  81181 tctacaagag ataagactct cactctggac aatcttagaa gatttgaggc tcagtgtatg  81241 cttctggagc tgggagttag aatccctaag ttcatggttt tcttctatca gtttgtccag  81301 tgaacttagg agcaaccaac caacttcgtt atattccttg gtcctctata tatggtcaaa  81361 ggtattatgt atagagtcat taaactcctt gcctctcatg agtggtgaat caggagtact  81421 gaatgcattt cttttgcata agtgtttgaa cagttcgtgc caaggactat cagtgttctc  81481 cacacaatta gaagtagagt ccttagcatt ttggggtcta atcatattaa gcaaccaact  81541 ccagaaaccc aaaaaccaac taaagaaatc tatccttctg taatcccagc actttgggag  81601 gccaaggcag gcggatcacg aggtcaggag actgagacca cagtgaaacc ccatctctgc  81661 taaaaataca aaaaattagc caggcatggt ggcgggcacc tgtagtccca gctactcggg  81721 aggctgaggc aggagaatgg catgaatcca ggaggcggag cttgcagtga gctgagattg  81781 cgccactgca ctccagcccg ggtgacagag tgagactcca gctcaaaaac aaaaaaaaag  81841 aaaaaaaaga aatccatcct taaaattctg ttcctctaga accattccca gtaccaaaat  81901 ctgattaaaa aaaaaaaaca ggcagaggaa ggtggaagga ctagatcttt ctccagtgct  81961 ggatgcttcc tgccctggaa catcagactc caagttcttc agcttttgga ctcttggact  82021 tacaacagta atttgccagg ggctctttgg cacttggcca cagactgcag gctgcactat  82081 cagcttccct atttttgagg ttttgggact cagactggct tcctgactcc tcagcttgca  82141 gatgcctatt gtgggacttg gtatcttgtg attgtgtgag tcaactctcc taataaactc  82201 cccttcatat attcatctat cctattagtt ctgtaccttt agagaacact gactaatgcc  82261 gtggggttaa gccaatcgtc tagtaggttc ataaaatgct aaaagcagat ataaacttaa  82321 agacgatcca gctcatctct tcattttaca gatgtaaaaa cagaagtaga aatagaattg  82381 gaaccccagt ttcttgaaac ccagtgtttg cataatacca tgctaatttc attctaattt  82441 gtgttttatt taataatcag gaaacagttc aaatagaggc ccagggctct gaaacattgc  82501 ccaaggtcta tggcttttgc aaagcaagta ctgtttctgc tactttacct agttgctctt  82561 ggcctatgtt tggggtgcat ctaaagaact gtttgctgat tattaaataa aacacaaatt  82621 agaatgaaat tagcatggtg ttataaaaac acagggtttc aagaaactgg ggttccagtt  82681 ctatttctac ctctaggata cagggtaagt cacttccttt ctcagattct gtttctttgt  82741 atgtcaaagg gctggattag ataaacttga cttcccctct tggccctcat gtatcattct  82801 ataaatatgt tatcatttct aatagactgt ttgatgtaat cttttgtcta atggcccctg  82861 cttttcacaa taaatgaaac aaaggtcaca agacttttat tcatttgcaa ccctgattaa  82921 ctaacagtta atgtgtaact ggagtgccac atagaaacag aaaggagaag gggaatggat  82981 tgggttggag aaggtgaagt ctgacctatc ctctacaaga ggtacagggt ttatccagga  83041 agacaggagt gccatgagga gtaaactcca gcaggggctg gagtcatggc cacacacagg  83101 gccttacagg atccaggctg cccagagcag agtttggatg ggggctggga ggctggggga  83161 gctagctgag gagatggttg tcatgccagg ccacgagtgt ggatgagtgc tggccagggg  83221 aggtatagga gattgatggg gctgtggcaa gcagggcaga tacccttcca ggaacctgtt  83281 tcattaaaca cagaatacag tcctgagggc tcggtctcaa tacagtcctg agggctcggt  83341 ctcaggaaaa catgttctta agttttacat ccttcttctg ttttgattag gtgtttcctg  83401 attataaaat aaattcctag aactgttaat ggtaacaaca caaagcacta tagacccata  83461 cgaggatcat attgaaacca atgacattta agaataaaag atctgatgaa tatgaacact  83521 tccctggttc ctaggaggat aacagttgag ttttgctcag gtaatctgcc ttctcttcct  83581 tcttctctgc tttRtatctc agtttctata ttttgtctta cattaatagg atttgttctc  83641 attaccctga tgattttatg gtaaactacc tcaaattctt tttggaagaa gatgggatat  83701 aaattatttt taaagtttat ctgaatagat gttcttcaat atcacacaat aaataatgac  83781 aatgtaactt tgtgcatata gcactttaag agattgatca taaacacaat cctattaatc  83821 ttaattcaag ttaataatgc ttgcatttgt atggcatttt aggtattata aagttttttt  83881 taattgtggt aagatatgca taaaatttac tatttaaacc tttttgaagt gtatagttca  83941 gtagtgttaa gtacattcac aatgttgtgt aaccatcacc actagccatt tccagacttt  84001 ttcatcatcc caaactgaat ctctgtacct attaaacatg acctcattct ccacctcccc  84061 acagctcctg ggaacctcta ttctactttc tgtatgaatt ttcctattct aggtgtctca  84121 tataagtgga atcatacaat attcatcctt tggtgtctgg cttatttcac gtagcgtaat  84181 gctttcaagg ttcattcatg ttgtagcatt tatcagaatt tgattcattt ttaaggctga  84241 atatcttcca ttttatgtgt ctaccacatt ttgcttatcc attcttctgt tgatgaacac  84301 ctgggttgtt ttcacctttt ggctattgtg aataatgctg ctatgaacac tgatgtgcaa  84361 gtacctgtct gagtctctgc tttcaatttt gggcatatac ctagaagtgg gattgctgga  84421 tcataggatc attctatttt taactttttt gaggaattgc cataccaccc gctacagcag  84481 ccgcatcttt tatattgcca acagtgcaca aaggctctga tttctccact ttctggtcaa  84541 catttagatt atcattcttt ttttaaaaaa acgtaataSc taacccaatg ggcatgaagt  84601 aaggttgttt ttgttttttg tttttaatgt gtgcgttatc ttacttgatc cattaaatcc  84661 ctaacaagaa ctcctctagg gcagatgtcc tgtcttattc atccttggcc ccagtgtctt  84721 gcaagcaagt gaattctcaa taagtgttaa ttgaatggat ggttgataga tttataggat  84781 gcatgccaat tctgtggacg agagtaggta ctaattgtta ttttcatttc acagaagagc  84841 aagtcagggc tccgagcact aagtgacttg gctgaggtca aactgcctgc aagttttatc  84901 taatagtgac agaggaacca atgtgtcgag catgaatgtc agtccattga aacagtgccc  84961 acttttctga ctctgctcct taagagacag ggcctgtaca gcaaggacac agagaagcag  85021 gttacagaaa aagggctggc tcatccgtgt atgcctggca tttgaggaat gtggctgaaa  85081 tctcaacact ctggttcaga agcacatctg caatcaaata taacaagaca tggtatgaga  85141 gatgtctggc ataccaagga gattcctaga atacagcgga taggaaaacc tcattacttc  85201 aattcccaag aaagagtact actggtagta atcccaacag gagtatccaa gtaactcggt  85261 aatcttcagt aaagaaaaga aattgtgaaa caattatagt cagtcctcca tctctgtagg  85321 ttccacatct gtggattcaa ctcacctcat attgaaaata ttaataaata aataaataat  85381 aacaatataa caattaaaat aatgcaaatt ttaaaagcat aacaactatt tacatagtat  85441 ttatattgta ttaggtatta taagtaatcc agagattatt taaaacatat gggagagtgt  85501 gcataggtta tatgcaaata ctacaccatt ttacataaga gacttgggca tctgtggact  85561 ttgttatctg caagggtcct ggaaccaatt ccccatggat actgagggtc aactgtacca  85621 agYtgaaaac aaaacaaaag gaaatctatt gcaaaaagga aattcccaaa gataaaaagc  85681 atgtctatga aatagtcaaa gagccgaaaa tgttgggaag acccacactg ctttccctgc  85741 cctgtgcccc tttttgcaga tctatttgtg tctttatcta ccagcagata ttctattatt  85801 ctaatagatt cctgtttttc ccaagagggt ttacttattt attaaatatg atgcaaacat  85861 ctctctcaga gatgttgctc tctcttccta ggattcctta gccctaaatc tgcagagcca  85921 attaggtatt aatattgggc tttacaactt tggaccatct gaccaccaag actgattcat  85981 taaatgtata tggtttcaga tcacattaat ttatcataga ctttagaatt ggctttttag  86041 aagtactgct ataaggaaaa tctcagcata gcaagtttta tctaatagtc tacttgtatt  86101 aaagagtact caatgtaaac cctaggaaga cttttaactg ccttttggaa attggttgag  86161 tgggatttga accgtgtact ctctgtaaag caggaaatta tcacttagta attactaagt  86221 atttaaaaat gggaaataga aaattacatt tcagacctgg tgcagtggct catacctgta  86281 atcctaacac tttggcaggt ggaggcaggt ggatcgcttg agcccaggag ttcaagacca  86341 gcctgggcaa catagtgagc aactccattt ctactaaaaa taaaaaaaat tatttgggcg  86401 tgatggggtg cacctgtagt ccccactact caggaggctg agacaggaag attgcttgag  86461 cctagaaggt caagcctgag tgagcagtga ccggccattg tattccagcc tgagcaacac  86521 agcgagaccc tgtctcaaaa aaaatgtata tttttaaaaa aagaaaatta cattttagtt  86581 gttccataaa tatcagtaca accaaaccta agtgcaaaat tcccaacaca aatgatcgcc  86641 tctgggccag ccatgtagcc cccatcttgc tgtcaataat catttccagg ggctgtaatt  86701 gtcttccttc cccctgtacc accccatcac aggacacaaa ttgttttgct tagtttagat  86761 agctgtgact aaactaagtg ccaattgtct tttaaaatat gtgttaatca gcactcaaga  86821 ttgttcctac aaaaatgtca ctccctcact caatttgcat gtgccctcct gagtaggaga  86881 gaaagagctg agttagaggc agccctctgg gacctgcaca gaaccctgta tgcttccggg  86941 agtgtggagt gtgtgtctga tctgctgctg ggaaaaggag aagaaatgct gagacactca  87001 ctgccagggg ctggtatcag gccattttca caggggctgt tggagggttg acagcacagc  87061 tctactggac cagggagggt cccagccagc agggcctgcc cctccgaaac tgtccctgtc  87121 cctgtccctg agaggcccca ctgagtgtca gatggcacat aagagatttc cttatgcgtt  87181 ggtgtgaagg agatgatcag ttccaggagg cccctccccc atgcagaaga gaagaaaatg  87241 gaagaaaccg ggttctcaga gtggcctgcg ggtgagtgcc gccttgtgct gtcagttccc  87301 ttcaccttcc agttctgggt gtacctagtg tggtttcatc aaactgctga ggccctggaa  87361 ttgagggagg atgctgaagg gtgccaggcc atagaagcag tagcaggagc tgcagcaaag  87421 agctagtgtg tctccagcgt cagcttttgg gccttggtgg catcaaagga gacgtacaca  87481 gggccactgt actacggatg ggattcttgg aacaaaagtt tgaataattg atgagtgtgg  87541 aagtgctatg caaaaattca attcaacctg cagttaccag gcattcattc tgtgacaggc  87601 attattgtga gggtggcaag gagacgggga gtctctgaga gactcagaga agagcagggc  87661 atggtgtcca cccacaagag atctgaagac tttcaaagta gttgggaaaa cagacataca  87721 acttttttcc cctccccaca gctagttgag gcaaaagaca tataacttat acagtgacaa  87781 aatactactg actgttactg acacataaca gcctgggctg ggtcactggt gagaaaaagg  87841 aggattcttg cttttggggc attcaagtcc tgtccattaa ccatttccca atcccatatt  87901 tatacatccc attttagaca tattaagctg aagattgatg tgacataccc aaagacactt  87961 aaggttgaag ctgagaatct gggctagaaa tataaatcag gatgggctgg gagcggtggt  88021 tcacgcctgt agtcccagca ctttgggagg ccgaggcggg cggatcactt gaagtcagga  88081 gttcaagacc tgcctggcca acatggtgaa accccatctc caccaaaaat ataaaaaatt  88141 agccaggtgt ggtggtgcat gactgtaatc ccagctactc gggaggctga ggcaggagag  88201 tcacttgaac ccgggaggca gaggttacag tgaaaggaga tcgtgccact gcactccagc  88261 ctgggtgaca gagcgatact tcgtttcaaa aaaagaaaaa aaaaaaaaag aaagaaatgt  88321 aagtcaagat taaagacaat gggtgagatc agcaaggagc atgtgtgcgg agaagagaac  88381 accaaggaag gctgcgtgtg gtgggaggtg gccggggggc agagaaagag gcggcggagc  88441 caaggagata gggcatcgtc tgaatggtga tgctgtatca acagatgtga aattcccaga  88501 ggtgtgaaac acagcagatc ctttacagca cgatgacagg acacagcatg agcctacctc  88561 tgccaggtga gaggagcttt ctgcaacctg tgatgggctc aaggatgctg accattcatc  88621 cgcccattgg gatagcccag gctctgagct cagcactcca cccgtcactg cgattgcact  88681 aatcctcacc cacccttgca ggcaggtatt actgtgggca cagagaagtt tgctaacttg  88741 ccagagatcc tgtgtaggaa gccaggtcag gaaacaacca gagtctctct aacagcccag  88801 gccttgaatg aacaccggcc tgcttcagaa tacatggccc gtgatgtgtt tgaattcaca  88861 gattcactgg acaggttccc tatagggcct gtgaggaaat ctggtctaac agaatccaga  88921 aagacaaatt tcgctaaaca ggtcaagcct gatactgctg ctacacttca gctgtgttaa  88981 gccactcggt tatcagacct gcttctcctt tcatgatttt agtaacacag gcctcttcct  89041 tgggcccctg ttgctcccac catctcccag gttctcttgt actcagggtt tagtctcccc  89101 tccccactac taatgatgca tgtgccttac tctctgatca tctctcctaa atctgctact  89161 cctctgctct ttctgtgcct tatctatccc tgccgagtct aacatgcaga ttttggtcat  89221 tccaaagtca tgtatagatc taatcacagg gctctctgct taccagctgc tacttggata  89281 aggaaagcat gccaacacgg tcctccttct tcatgctggc caagtcagca tcattattat  89341 tacctaagtt tatttctaac acatctcaat atcttcatgc actccctctt gataaaagta  89401 actgagcata gcaccatcaa taccatcaaa tctgtcattc tcttcccctc tctctgggtg  89461 ggacaggaag ccaggctgct ctaggaaatc ttccctaaca agcaaagggg acttgcctgt  89521 cctctcgcat gtgtgatctg agcttgtgtg gatcccagag tgggcattct ggactacttg  89581 accttgccta tctctccttc acaccctctc atctctccct cctaccacca aaaaacttgc  89641 atcgtatttc caatctccag acatactttt gaaaccattt atctatctgg tgtgtttatc  89701 tgtatctagt atgatgtgaa tatgtgattt gtatgtgtgt ctaccactgt tttggtcagt  89761 ttgtatttct cgtgggtaca gttctatgtg ctcatgtatg tggaacatat gtatactgac  89821 acatggacct agctccaaat gatctgaaag gaatataatt gtaattgaat atttgcacag  89881 atatacaaca tacacatgtg atggctgggg gaaatgcatg tgggatttca gtcagcattt  89941 tattagagaa ggtatgtcat tagtgctgta ttaacaatga atcagcttat ttgtgggtca  90001 ctgtcaatga ctcctttgca aatcacacat gtaaatattt ctgtctgtgg tctgatgaac  90061 atgcagtgcc acagtctggg agatgctgag ccatgccctg tgtaggcagc atatgaaaag  90121 aactgcatga tttaaaagat gctgaccagc ttaaggaaag caatttaaaa acttcctaaa  90181 aatctagttt gaatgaacaa cagttttcat tttgtgtgtg tgttttttct tttagagatg  90241 gggtcttgct atgttggcca cgctggtctc taactcctgg gatcaagcaa tcctcctgcg  90301 gctcagcctc ccaaagtgat gggattacag gtgtgagcca ccgtgtctgc tggccccact  90361 gttttaaacc ctgattcgac aatcatacat ttaactcatt acctgtcttg ttccttttac  90421 gacaaactca aagcttttat tttactaaag tatttgggtt aatcttttgc ttttctgtca  90481 tgttctgaaa tatgtacaat aacagaatcg ctcaaaatat tatctccgtt aaatgttttg  90541 tggctttagg gagaggtcta acaacatgcg ggaaacaaga aatcaagcgc atccaggatt  90601 catttataat ctctctcgtt gagtagaagt ccgcatctct cgatattgtc tggttacctg  90661 catgaagtat tctaaaggag gaaaatagct caaagaggac attcatgtgc actctggctt  90721 ccagttggcc atttgagtaa gtgatcgcaa ttaactgacg agcggcaggg aaacacttcc  90781 tggaattctc atctacagac aagaacaaac tggggcgggg cccatcacct tcacctacgc  90841 gccgggaggg tggcggctgg cgggcggggc cgggctcggg ccgtgacgcc gagagtgcgg  90901 ggcgcgcggc tgggagcctc gcgcccccgc ccgggcccgc ccccatcccg cccgcataca  90961 gcccgcatcc cgccggggaa gcgagcccag tccagcgctg cccgtccagt cctcgcccaa  91021 gatttaaagc ccgcaagttt tgttcttgag accagcgact ttagctccga tgcgggaagg  91081 aaagccgacc tccgatttgg acatttaaag agctgggctt gaacttcgtg agtttcgctc  91141 taaactgccc ttgaaatgaa gctggacttg gaggtaaagt cactgggaag ctggcctggg  91201 gcggggtttc cccctcttct cgtattttag aaacggacag cggcagtgca gccctagttt  91261 gctgtaagtt tccttacttt gttactgagg cccccagagc tccacgcata agtggtgtga  91321 ccagaaacct tttaacaaga cccgcctgag cctgcgttag agctcccgct cggaaagtaa  91381 aagaccatcc taatccgcgg cgctgcggaa ccggtgtccc gtgtgggagg aaccgcggcg  91441 ttccctgggc gtagggcccg cgaggccagc acagtccgcc tcttggcgga gcgccctggg  91501 ccggtggttc cgcgcggagt tagtctgtgg tcagttacgt ggtgaaaaca cggctgtgcc  91561 gcggccgcat ctttccgcgg ccgaggcctc tctgggtggg agtgttggct tcctttccgg  91621 atcgctaaat ggggaaagtt ctggccgctc ggcgggatac gtctccaggc cacggatggt  91681 tcgttctccg tgccgcggcc ccgagctggg ctccctgggt ctccagcgcg ggctcccggc  91741 attgggggct gcgggccggc ccctccgccc cgcccccgcc ccgccgcgcc tcctcggccg  91801 agcggctcgc ggtctccggc gcgggaggct ccgagtctgc ccactccggg ccgagcgagg  91861 tctctggagg agaagagtgg cgaggaggtg agggcacgcc ggccctcgcc cggcgggtgg  91921 cgccaggact tcaggtggga acgcgcgctt gggccggggg cgcgtggctg gcgtggacac  91981 cggatcgggg cccgccgccc tggcccggac cgcgcacggc ccagcgccgg gaagtcggga  92041 agccggggag gcctctccca ccgcgggccc cggcagcccg ccctctgaaa gcgcggcgga  92101 gaaggaggct cgtcccctcc ccggaacgcc tttgttccct ccggcctgcc cgcgcgggtg  92161 gccagcggct gggacccagg ccgggccgcc gcccaggtgc ggcaggtagg ctcgggggcc  92221 gggcagctcc ggttggggcg gcttcccggg gcctgcgggt ccccgtccct gaggagctcc  92281 ggctcctcgg tggcgggaca ggcccgtgcg cgggagccgc gaggcgaacg ccgcgcccac  92341 caattcggtt gccggccggg ggccccaggc ttgcggccac ccgcctccgg ctggagggct  92401 gaattcgagt cgaaagcccg tgtcgggctg gaaagaagaa accgccaacc tgagaacgct  92461 ttcggcgagt tactggcggg ggaaatgggg acagggaagt gggcaggcgg ggagactgca  92521 gccgcagatc tccctggcgg ggaggtcgtg gccactcttt cctttgactc tgcctcattt  92581 cattttgaat cctgatgtga cagaggcaat tgcttgcttg gataccatat aggtaaaagt  92641 aacagttttc aactcgactc ttgactacac cctgtacatt cttggccggt gttggttttc  92701 ttaggttatg gatcatgtta aaggtacacc gatgtggtaa ccgcacagtg gccgatggtg  92761 gcctgaggct catatttatg gtaattatct gatgaaagta catccatcag aattggattt  92821 gtgcgtctgt gcctttattt tgggaaacct tgcctgttcc ctgtggggga tgggagagga  92881 atgtgaaaag ccgaagttga cccagaaaag gatgattgaa ggtagttgta ttaagtggtg  92941 gcaccgaaat gattccaccc tgaactttct gaaagggtga ttagtgatca gcagcagacg  93001 ttataacttt ctcaaaataa atttatggta gattttctat ctggtcacaa gtgaggaggt  93061 gaaagctgct ttttggggcc aactctttgc ttttaaagca agctaaacgc aataccagaa  93121 aggtttccat tctgtactta acctgcttgt ccttctcact cttccttatc ctcccgcaca  93181 cgctctgagc attgactgag cactctgagg aggaggctga ctcaggctga cccttcccgg  93241 ccctgcaagg ctctaaggta gaaccagtgt tatcacagga aaggcccaag ccaaagctca  93301 gagcagctgt tcctgagaaa gtgggagatg aggatgagta ggaggtagag atgcttataa  93361 ctgctctgcc cagagaaaac tcagaaggtg cagaagagtt ttcaaaataa agtgcaggcc  93421 ccatcaagta aaaaatgaaa cattgtattt cattaaaatg gtgatcagtt ttctcttttc  93481 ataaaagaca ttcaaatggt gagattgtaa aaaaaaaaaa aaagaaaaag aaaactttct  93541 aattctcaaa aatagaacag cttgaataat aactcatggg ttttgaaatg ggtcatcttt  93601 taaaatgggt cactttggct agaatcattg cacttggtga ctttcacatt gtaaagggtc  93661 cagttctttc tgaggcccaa ttgcactaga atctgcattt cagaacacag gaggtatcag  93721 gaagaaactg ggagaaattc agagggaaat tgtctcttct cagagtaaca aaatgtcccc  93781 tattcagggg gaattttata tatgctgaag taaatagatc tcagctttga atttttataa  93841 tacattatgc cttttctgaa atatttccat agccattatt ttaatgcatt gttagaataa  93901 ccttgtaagg agaggaattc attgtatctg tgttccagaa actgaggcac agtaaattac  93961 aagagctctg ccctttgata tcgaatctct gtgagcgaag atgtaaccag aacagtgtaa  94021 atctagaatt actttcctta cggtatgcaa tatctaatta ttatgtatta gtttacatat  94081 atatagggga tgtaagaatc tttatattta aaggataaga aacttgatca ggtgagatac  94141 aagatttgaa taaaaaagcg attttttaga aacattttaa ttctacaaat tcagttgctg  94201 gctttgatat gtaacttatt cagatttctc ttccatagaa ggctttaaca ctagaagcca  94261 gttctctaca aaagagaatg ttctaccaag tgtgtaggca acaaagccca aaagttcaag  94321 ttcaaaatac tgttctacag cctaagatcc acctaatatt tcagatttga ctcttttgct  94381 cctttcctat aacttcctat aactttttca tagtgctcta agcaaagtaa gttcaaggat  94441 ggactcacct caagtttcct tctcttactt tagaggacca tataatctta tacctgtgtc  94501 atcctaattg aaatgtattt taagtgcttg tgggaacaag aataacggaa gaccgttatt  94561 ccattaatgg aataatgggg aacatgagaa ggaccatatt cagtcaatta gggatgatac  94621 ttctgcctca acagtatttg cgatgtgtta aatggccctt agccatcaga tcagtatttt  94681 taaaaactcc tacctaatta atgttttttg agaagaagca tattatgagt atagctcagt  94741 attctaaaaa gaaaaaaacc tgaaaaaaaa atcagccatc cattaactaa cccatcctgg  94801 taaagctaca caaacagatt ctagagaaag gaaatttgca cagatggaac atctaatctg  94861 gactgacatt gtcaacaagt tatctcaaat gatcctgaga aaacactggt acatggttta  94921 gagacaagtc gagctcatgt gactagtaaa tggagaggca cagtatacat ctacttttgt  94981 tggcatttaa acactctcgg cttttttact ctctttcacc accatcaggt ccagattcca  95041 gcttgcactg gaatatttat tgaccctttg atagaagagc tacacctgaa acatctctga  95101 agtcttttta gcatcctatc ctgcccatgg cctactacac agtatgtgat aagttaatgt  95161 ttgttgaatt agttaaatat acgtattaac ctactttcag ggctgcccgc tgtaatgcct  95221 agaatagggc aggcacttaa taaatggtag ttaacttaga ctaaatgttg atccaccaag  95281 acagaaacat gttacatata cctcttgttt tacaccatct tctacttttt gtacctctgt  95341 gttataaacc ctctatcaag tgttgcttct aaagacaatg tccagaatgg gacgtaggaa  95401 ctgccactgg gcaacaggca gacactgggt tatatcgtca tcctgtttct gaatcctggt  95461 ttttcttatt attattcaac cacagctaaa ctttctgatc tccttttttc cgttttttcc  95521 ctttacccta acttctagcc aaactaagtg atttgacatt tccccactca agccacaact  95581 ttttctctta tgtgcttaaa aaaaattcct gcgtagtctc tcttggaatg tcttattcct  95641 tgctccctcc acttcaccct tttttttttt tttttttttt ttttttgaga cggagtctcg  95701 ctctgttgcc caggctggag tgcaatggtg ccatctcggc tcaccgcaac ctccgcctcc  95761 tgggttcaag cgattctcct gcttcagcct cccgagtagc tgggattaca ggcatgctcc  95821 accacaccca gctaattttt gtgtttttag tagagatggg gtttcaccat gttagccagg  95881 atggtcttga tctcccgacc tcctgatccg cccgcctcgg cctcccaaag tgctgggatt  95941 acaggcatga gccaccgcac ccggcccact tcacccttta agccagttga aatgctcctt  96001 gccctactcc tcccttctcc ttttctttct cccatccaag tactaaccag gcccaaccct  96061 gcttagcttc tgagatcagc caggatcagg tgcattcagc ggggtatggc tgtagacttc  96121 tctttctgag ctgtgtcttc ctcctttcct ccccaaaccc acatttgaat tctgttctag  96181 ctcctgctca tttgccacct ctcccacaaa gccttcccca gtttccatct tctccctgct  96241 tccctcatct cagaggaaag ctcttcattc acagagcacc tggtttgggg tccatagttt  96301 gttgtccctc agtatccttg gggaattagt cccaggacac cacccccccg cccctgatat  96361 ccatgcatgc tcaagtccct tatataaaat ggtacagcat ttatattata acctatgcaa  96421 tattcccatg tactcttttt ttttattttt atgtgtttag agatgggtct cactctgttg  96481 tccaggctag agtgcagagg catgtagcct tgagctcctg agtcaaatga tcctcctgcc  96541 tccgcctccc aagtagttgg gattacaagt atgagctacc acacctggcc catgtacttt  96601 aaatcatctc tagattactt ataataacta atgcaatata aatgctatgt aaacaattgt  96661 tacacggtat tgtttagtgc ataatgacaa gaaaaaaatg tgcatgttca gtacagacac  96721 gaccataaat ttttttttca aatgtttttg atctgtagtt ggttgaatct gaggatgcgg  96781 aacccatgaa cgtacacgac caactgcata tgggtttcta ataacagatt tgtggatgaa  96841 gcccacccaa cataatgcaa catttcaaat gtatcttagt cagtttttct ggggcaaagc  96901 cagcctaaca aatctcaaga atgtttgaaa aattctagct tgagtgaacg ttgttcttct  96961 agtgactcaa ataaagcagt cattgtgtct gaggagtcct agtggggtgg caggggtggg  97021 gctaggagta gtggctgttt gtagcatgtt ttatatcaca ttaaagggct gggtatgcat  97081 tttaggcaat aattcttact cttattgata ggagaacttg tattttttat tcatctacct  97141 tatgatggat aactggttta catctcttca tcaggtttac ttcttacgtg tcattttatg  97201 agctgacttt gaatatatct tcagattttc cctgccttta cacatgaaaa cggttttgat  97261 acagcttttt cattagagaa tggcagcttt tccaggccag ggtgtctgtg tctgactcct  97321 cgcctttcat tttacccaat acttgcttaa aacatcctcc ctgagtccgg tacttttttg  97381 cctcctctcc ttccttccac cccacctctt tttgtcaccc tcctgcgact ttggatctcc  97441 atccttggag tccctgaatc ttttttgtgg ttgaaagact acacccaaag gacacagact  97501 gatgaggtca cttctgccca ctgtctagta ctagtgagac caagaagaag aaggagggaa  97561 aactgaaatg gaaggtttga aaaagagtgt gttagtgaag gaagtaagaa cagtgctaac  97621 agaatgcagc aaaacagaaa tgacttcaca cttctggcag tgaagcaaat tctcatccat  97681 cttgggttgc ttctcttgac cctgcccacc tcctcccctg ccctcccctc ccgatgagaa  97741 ctgcaggaac acctgtgccc atgccaagga aaaagggcag gtgaggaaca cagggatggg  97801 gctgggactc acaagctgct ctaagctgcc acagggtctg gaatttgttg acattacatg  97861 gttaactcgg tatctcgatg cctcagtatc ttctgcaaaa tgtggaggga gatcctttct  97921 tcctcaaagg ttgttgtaag gcttaaagag ctaccacatt cgtaaggcat ttggaacagg  97981 gcctggtata tagtagacat acccatggta gtaactgttg ctagactggg taaaaggaga  98041 agtttcttaa gtaggcaaga aaaaaactga caacagttat atttaaaaga gcaagcaacc  98101 gtgtcagccc aaacaagtgt gttgcaagaa tgagcacaga gggccaagct cagaaaagcg  98161 agtgtttgac ctctctgaga cctcctttat atgtcagctt ttttatttgt ttcaaatttt  98221 gcataatcaa actggttact gggaattatt gagttcatat aacttcccta aaatctaaga  98281 ataatctata atcccatgcc accagtcccg ttaataaaga cttagggtct gtctttaccc  98341 atttgttcat ttgaaattcc ccgtctttac ttcccccata accaggaaac agatctacac  98401 ccatgtttaa cttataaaga gatataagtg agtttacata ggtggagatc ttgtacctgg  98461 attcacattg taggttttat tacccagctt tcccctctat cccaaagcca tgattgccat  98521 agtggaattt aaagtctttg gactaacact gaatcaagaa ctaactctac aaatgtattc  98581 tctgcaggag gataagaatt ccaaatggct tctaattgtt gcccatggct tgaaataaag  98641 ttccatatag ctaaagcccc agcacaaaaa cacttgaaga cagccattag gtagaatttc  98701 ttttcttttt tagaggtctt atattggaaa tgtaacagtt gcaaagatat ctaatgtttc  98761 accttaaaat gtgagttata aattcacatt tcacattgcc tccttcctcc ttgagcaaac  98821 agactaggca attagtacca aatagtaact agttatattt cttaacaccg attattgtaa  98881 gtaatttatt aagaaaaaat ctaggaagat caaattatag cttttccagt aaatctgggg  98941 ctttttattc ctgatctctc tcaacaaggc agtttggctt ctgaagattg gtcctagcta  99001 tacttaacag gaaacagatg acgagaagaa gccaggaaaa aatacttgga atatagaaca  99061 aaatgcagca taaagatggt acagaagatt ttatctttct ttcctttagt tatgtgtgga  99121 caggaactaa aaactctccc acgtgggact ttattaagta ataaaagttt aaatgtaaaa  99181 gatataaagg aatgaggata taaaggaaat ttttttaaaa actgtgactc accggaggag  99241 ccttatatag tctactttct aagcatatca cttttaaagg gatcattata ttttcatgat  99301 gcatggggat attccaagta ttaaattgat tttttaaatg aagaaaatta tcagttcagt  99361 cttgttttct atgtaggttt cactacagta ttttatttcc cccgcaagga aaaaaccaga  99421 gctagaatgg aatgattaaa tttcttagtt ttcttctaat ctgttagtct ttccattaaa  99481 ttatactgcc ttcttatata gaatttctgg gtttgttctt gttttggcaa taattgaagt  99541 aagagaggat gattttatgg tagaggtttg attccattct agattggtgt ttttcagcta  99601 tgtagaacac aatccattca tggttcagac aaccgtttta ggaggttttc atgtccagca  99661 ttaaaaaaaa aaaaaaaaga atagaattgg gcattcatat atatatatat atatgaatat  99721 ttattaataa aagtttaaaa gatataaagg aatgaggaat gcatatatat tatatatata  99781 tataccaaat taaagtatca taatactaat acaaggatta gtatcgtttt atgatacttt  99841 attatataca ctaagggtta gtcttgtttt atgatacttt attatataca ctaagggtta  99901 gtcttgtttt atgatacttt attatataca ctaagggtta gtcttgtttt atgatacttt  99961 aggttggtat acatacatat actaagaatg cagacatgtc catggaagtt ggcaatattc 100021 agcccccagc aggccccagt cagcaggaca gggattaggg gagtcaggtg aggcagggct 100081 gtataagtgc agggttggag cctgtcttgg tttaccgttt tgacattttg ttcactgtgg 100141 atttttttca gtaactttga ttttttaaaa atattgcatc aaaatattat actgactacg 100201 gagtttttgg taccccctta aattgtgcac ctaaaataag tggctccctt gtctccccct 100261 agtcccggcc cgatcagtat gttttataga gggaatggtg agtgttgggg aactagggag 100321 cccatgagga ggcggcccag aaggggagga ggagctgatg gtgtgtctga tgtctcctcc 100381 tttctctagg aagcagacag gaggtgaagt cttcagggtg ggggggacag agcctgggag 100441 ggggagattt agcatggccg ctgaagagac tgggaaaggg agaaggttcg ggacaagttc 100501 aggaacagtc acgtagcact gaaaactgct caaagctaga tagtgccatt tgtggtgacg 100561 ctgcctgcct ggtaagtccc atttccccag cagccctgga ggtgtcatag gaacaggaag 100621 atgggagggc gctggggtct ggggttgttg gggtgggagg gcaaaggtgt ccaatgattc 100681 tgcatctgaa acgggggagg tgtgtgagga agaaaggtca ggagactgct gggcagggtg 100741 gggtggcaga aggttctcga tgaatattta gaagttgcag ctgaggagtg agtgtactat 100801 agagtgagta tcctggaatt tgaggtctca gaagaaatgc agttctcagt gaagccatgt 100861 aaacaacttc ctggctgata aggtgtcttg agtgttggga gatcacatga tcaaaatggt 100921 atacacacca taaggtgtcc ttagacttga gtgtggtgta agtctactga ataaaatgca 100981 gaaatacagt tcactgttgc ttttcttcct ggaaattgcc ctccccaacc tcaaaccagc 101041 ccccaagtgg ggcagggtcc atctttagac cctcctgtag cacccagcac aaccctaact 101101 atatggtcaa tgtctctctt ccccacttag agctccaaga ccagggcctt gtttgtcctg 101161 cttacccact ctacccattt gttaacccca tcttaatttt ctgatgaatg agcccttttt 101221 tcattgactc ctcctttctc tccttctccg gagtttattt ttcctccagg cccattgctg 101281 tctctccacc tagagagctt atccacattc atgaaattag tcatcttcat gctcagagta 101341 cccacctgga tctctccagt cctgtctaac tcgtcttaga ttattacccc ctggacatct 101401 gtaccttctt gtctctccat cgcctcgaac tcaatgcgag tctaaaatca aatactcctt 101461 cactcctaac tcaggaaacc agttttccct cccttgttgg taatgcttga aattagctct 101521 tacttctctt cttcctcctt tactcgccat cccaggtcat tcatcaggtc ctgctaggtc 101581 tttgattttc ttgaaagggc cttctttatg ccttccttcc gtccccattt ccaccaccct 101641 cgactaggcc ttcctcacct catccggcat cagtgtgagc ccttgagaaa ccagcgtcct 101701 gatcttcagg cattctgcat ttcaccccat accaccatta gccagtttaa tccctcttgc 101761 tcaaacttaa taaatactga gcaagggagt tactataaca aacaaaacca gaaatttcta 101821 tgacttcctg ctgctttctg taccccccta agactcctct acctgatttt caaggttctc 101881 cataagcagg ttcccacctc tctgctttca aatcttctat ctgccataca cagttcttgt 101941 ctaaaacaaa gattgttcct actcctgcag ccacctaaat cctagtcgtc ctcaggccct 102001 cctaaaatcc tgactaatcc aacccccacc gatttctcta ctagtcccag agacttccct 102061 cttgtgcctg gggttggtgt catgcagaca cactgtacct taccaggatt tgcccattgt 102121 tgtctgtggg aactatgctt tcatgtggga actacgagtc ttgtcacaaa tagagtgtaa 102181 atgctttgag attggagact gtaaaatact tcttcatgat ctcccactgt gtctggtata 102241 agaacccgca gagtaaatac tcatatactt gtaggattaa ttgaaacaac tggagactga 102301 ggttgtttaa cttgtactta gaagtgtgga cctccccatc ttgattaaaa cttcaaaacg 102361 gatagcaggt ttcacatatt atctcagcat cacaaaagta gactcaacaa agaaatgagt 102421 tgacagcagg ggtaaggttt acaacaatca gggattattg cagggaaact tgtagccttt 102481 gggtgttcta actagttttt tcccaaaaag tttaaagagc tctcttgtga aagaatattc 102541 ttatgagtaa ctgaggggca cggctcctag actgaaaagt atttgggatc tgtcacctct 102601 tttctgatgt tcctacttct attctttatt cttctgacta cctctattag aaaagataat 102661 actaagaata cctgtccctt cttctcagtc caaatagaag caaacccagg ttgtatctga 102721 gatatctgca tattttcttc taagcaattc tttgtcctct tctctcccat gctttttcta 102781 tttcctattt tcagcaggct ctgaagtcat ttataatttt tactgcccct cggtgacatt 102841 acatggatat tcatccctga tttgcagatt aaagcaccga atcagagtga ggtgagggtt 102901 gcccaaggtt acacaataaa tctggcccca aacaggggta acccttgagt ttctagtctg 102961 ttgattggcc actgacccgt gctgcaggca cacaaaggaa gctgcaccca cagcagtctg 103021 ttgtggatgg ttgctgagct gcgcattcgg cattgggctt gctttgtttc ctgccaggcc 103081 cagcattttc ttctaccaga tcggcaggct tgtgggcttc ttcctaggtc cctcccctgc 103141 actctgaata ggaaagctgg aagctgtgct ttagagaagc tttaagacgc cgaaagaaac 103201 cagaagagtg agcgccagtt gtatgtgcgt ggtctccatc cgcaaagccg gagctgggcg 103261 caacagtgtt gacttgtaat tgatcaattt agatcgggcg caggccgggg gagggcagtg 103321 cttttgattt aggctgggaa aggcctccta gtgactatgt tcaatttgga ggaattcaga 103381 tgttcttttg ttatacaagt gaagctgtgt aatacaaatg aggagtttta cttttcctaa 103441 atcttcccct tatcattcaa gtattgagga gttttacctt tcctaaatct tccccttatc 103501 attccagtat tatcagtgag atctggttgt gatttatgta aatggtggct aaaaaattca 103561 aactactgag ggggagaatt ctcattttac agcttcacat gctgtgctga actaaataag 103621 tagcgtggga tgttggcttt gtgacaggtc ttttgtcatt tttcagaaag cattttgact 103681 tgttgatgtc aatttggaac agctgaaaaa atacaggaaa ataagataaa tacgtacatg 103741 ttgagggtgg ggacaaaatg aaggttctga accagctgcc ggcttacagt agccatataa 103801 gcaacagcag caatgcacca acctggtgag taataggcct gattcactgg agagatacta 103861 gcacctttaa tgagtcagat agatgcacaa tgggtgtggg agcagttgga cttgtgggca 103921 caaagtctag caagaagctc agacttgcaa acaactgtag gacgtgcaaa gcaagctggc 103981 attggagctt gccgggcaca gctgctcagg aataggcagc tggttttccc tttgatccct 104041 gagattccaa aggttacttt cctctttgtt cccttcccag ggtcaattag agtagaaact 104101 gcagatgctt ttcagttgag aattttccta gaattctcaa aaatgtgtat gctggcttaa 104161 aatctgccat caagaattct gttaccttgc tttaagcctc cagttccttc cagatgtatg 104221 gtggaggagg ccagagggcc cttgttttgg ggcttcagag gatggttgtt atctggatga 104281 gcactgtgga aagactgaga gagcaactga gagaaagtgg gcccctgaat gaaagtgatt 104341 tcgcaaattt taggcagatg ccaccatcag aaactgatat tttctgacgt ctttctcacc 104401 ttcctctaga gcattcagtc cagaaatgac cagcctgtcc aaagggggaa attactgata 104461 ttgatctgtt ccttagagca gtgtttcagt cttttttttt tttttgagat ggaatctcat 104521 tctgtcaccc aggctggagt gcagtggcac gatctcggct cattgcaacc tccaccttcc 104581 tgattcaagt gattctcctg cctcagcctc ccaagaagct ggaattacag gtgtgcacca 104641 ccacacccgg ctaatttttg aattttttat agagatgggg tttcaccatg ttgccaggct 104701 ggtctcaaac tcctgacctc aagtgatcct cctgcctcgg cctcccaaag cgctaggatt 104761 acaggcgtga gccaccatgg ccggccttca gcctttgtga tattaaagca cagcaacaca 104821 tttcccatta cacccctgaa cacacacaca cagaaaaccc aaaagtttca caaaatgatt 104881 cttgctctta ctactctcag tacactctgt atttaaaaaa aaaaatgctg gttgtggctt 104941 cctaagtggt gcgtgcagtt ttcaaatcaa tgcccttggc gataaagtgt gccctatact 105001 gattatctct ggacaaagtc tgaatggggc ttggctctaa tctctagtcc tcattggaca 105061 ttttacatac ctggcctttg cctccaccct gatgtggagt gatcatgggg gtgggaaata 105121 tagctggatc cgaaagctct gaagtgggga tggaggtgtc acagctgagg ctaggcccat 105181 tctgcagggc actcagtgtg tacagttggt tttctatcag gggtcaaccg gcggggggac 105241 ttgagaacag atctctgggc acaaagcagg gcctttgccc tggggcttgc tatgtggctc 105301 agcctacacg gctctctccc cgtcagtcct gtccaaagcc caggaaacta atgtaccacc 105361 cccgaggaag agagcctacc tttccatcca aggaagtgtt ttacctgtgg taagcacggg 105421 ggacagaatt cttgaggaag gagggtgctg cgtcccagtg gtggaggaaa agagaggacc 105481 tggtgtaagc agccatggca tggacctcat ccgaggtggc acctggctag ggtcctgacc 105541 tccaatcctt ccccagtaac catcactttg agtaaacagt ggctccaccc ccggcatggt 105601 tctttgcacc aacatttggg gaatgcctac caggggtcac acactgagct ggatgctgag 105661 tgtagggtgt ccacaacatc gtgcctaaaa agtctctgta tggggtataa gaaggtgctg 105721 gggcaataca gatgagatga gaagcatctt tcagggaatg ggttgatccc aattcaggct 105781 tcccagagaa ggatgtctgt agacttcata ttagcaaggg aggaaggtag ccaggccaca 105841 ggactgctgg tgtaaagacc agggcatatg aaatggcaag tgtgactgtg ctttcagcca 105901 ataatttggt attgtcaaat gatgggacca aacagctgga gaggcagatc ctaaagggtc 105961 ctgtgggcca ggctggactt catcttgtca ctaactaatg gagaggctct gaaggagtta 106021 aaagagctca gtttgtctcg tggttaaatc caagttttac aaaggtcacg ctgactgtaa 106081 agtggaaggt gggctggcca ggggatcatc tagtctgggt gagaagtgat gataacatga 106141 aggggtgaag agagatttag aagaagtgat tcacaggatt aaacatttaa ataatggaag 106201 tggagaaaat ggggggggcg gttccagatt tcaggcatag atgaaagaag tgcagttagg 106261 cacatgtaaa gagaaacagg aacagcaggt tttaggggag aagataacag aatgggtgag 106321 aaatgacact tgagtaccct agtgtgctag gtaatcatct gtctacttcc cttcatttgt 106381 catgtatatt cccatttaat ttgcataaag acttcgagtt aaacggtctt accccaattt 106441 gtcaaatttc tgcgcatgat atggtacaag aaaccgtaag tggctaaggc ggcattggtg 106501 ttcaaattgc ctgactacaa aggcagtgct tgttggctac attctgttgc ttcccagttt 106561 agaacatgtt acattgaggc gcctgctgca tttccaaata aaaaagtaca gaaagaaggt 106621 ggctgtataa atctggggct cacaaagtaa ttttgattac tgagagtttg ctttcaagga 106681 gcaaactgtg actccttgat tatgaacctt aatttaaaaa aaaagaaaaa agaagtctta 106741 ctcttattcc tgccttgtct ggggcaagcc ttaatggatt tttactgctg tgaattttct 106801 tttcattgaa gattttgcct tgatctatgt atctgctttc atcctgacca tattcaagtc 106861 agtatattca tgaatgtacc tgtttgtgaa atttgaactt aagtatacac gattatagcc 106921 gtttgggaag cttttttttt ttttttttta agagtaggag tagaaaaagg tctctgtact 106981 ctgaatggga agacagtgta aagcaatttt ttcccttttc ctgtcctcct ttaaaaaaaa 107041 taaacagccg tatgcctctg ctaagtacta actacctcat caccttttgt gcagacaggg 107101 caggttacat ttggttttaa ggaattagga atatgtttct ttccagcacc ttagtaaccc 107161 acgcgattgt gattcttttc tcttcttgac tgtgataggt ggcatggaat attcacatgg 107221 gagagccgca tgaggccgcc caccacgctt cctgaaggat gcccgtgtgg aagaattttg 107281 acgtgccagt gtcctcgttc tacagggtgt tccattcttc cgcaatctca gaaaaatggg 107341 actaaaagaa actattttgt aaaataagaa gacttccatt tttaatgacc aacatgtatt 107401 aagatggaca cctactctac gaaacacgaa gttctatggt ctcgaagaag cccgtgcctg 107461 tttaaaactg atcctaacta aaaacagact tgagtggata tgagaatgtt ggttagtggc 107521 agaagagtca aaaaatggca gttaattatt cagttatttg ctacttgttt tttagcgagc 107581 ctcatgtttt tttgggaacc aatcgataat cacattgtga gccatatgaa gtcatattct 107641 tacagatacc tcataaatag ctatgacttt gtgaatgata ccctgtctct taagcacacc 107701 tcagcggggc ctcgctacca atacttgatt aaccacaagg aaaagtgtca agctcaagac 107761 gtcctccttt tactgtttgt aaaaactgct cctgaaaact atgatcgacg ttccggaatt 107821 agaaggacgt ggggcaatga aaattatgtt cggtctcagc tgaatgccaa catcaaaact 107881 ctgtttgcct taggaactcc taatccactg gagggagaag aactacaaag aaaactggct 107941 tgggaagatc aaaggtacaa tgatataatt cagcaagact ttgttgattc tttctacaat 108001 cttactctga aattacttat gcagttcagt tgggcaaata cctattgtcc acatgccaaa 108061 tttcttatga ctgctgatga tgacatattt attcacatgc caaatctgat tgagtacctt 108121 caaagtttag aacaaattgg tgttcaagac ttttggattg gtcgtgttca tcgtggtgcc 108181 cctcccatta gagataaaag cagcaaatac tacgtgtcct atgaaatgta ccagtggcca 108241 gcttaccctg actacacagc cggagctgcc tatgtaatct ccggtgatgt agctgccaaa 108301 gtctatgagg catcacagac actaaattca agtctttaca tagacgatgt gttcatgggc 108361 ctctgtgcca ataaaatagg gatagtaccg caggaccatg tgtttttttc tggagagggt 108421 aaaactcctt atcatccctg catctatgaa aaaatgatga catctcatgg acacttagaa 108481 gatctccagg acctttggaa gaatgctaca gatcctaaag taaaaaccat ttccaaaggt 108541 ttttttggtc aaatatactg cagattaatg aagataattc tcctttgtaa aattagctat 108601 gtggacacat acccttgtag ggctgcgttt atctaatagt acttgaatgt tgtatgtttt 108661 cactgtcact gagtcaaacc tggatgaaaa aaacctttaa atgttcgtct ataccctaag 108721 taaaatgagg acgaaagaca aatattttga aagcctagtc catcagaatg tttctttgat 108781 tctagaagct gtttaatatc acttatctac ttcattgcct aagttcattt caaagaattt 108841 gtatttagaa aaggtttata ttattagtga aaacaaaact aaagggaagt tcaagttctc 108901 atgtaatgcc acatatatac ttgaggtgta gagatgttat taagaagttt tgatgttaga 108961 ataattgctt ttggaaaata ccaaatgaac gtacagtaca acatttcaag gaaatgaata 109021 tattgttaga ccaggtaagc aagtttattt ttgttaaaga gcacttggtg gaggtagtag 109081 gggcagggaa aggtcagcat aggagagaaa gttcatgaat ctggtaaaac agtctcttgt 109141 tcttaagagg agatgtagaa aaatgtgtac aatgttatta taaacagaca aatcacgtct 109201 taccacatcc atgtagctac tggtgttaga gtcattaaaa tacctttttt tgcatctttt 109261 ttcaaagttt aatgtgaact tttagaaaag tgattaatgt tgccctaata ctttatatgt 109321 ttttaatgga ttttttttta agtattagaa aatgacacat aacacgggca gctggttgct 109381 catagggtcc ttctctaggg agaaaccatt gttaattcaa ataagctgat tttaatgacg 109441 ttttcaactg gtttttaaat attcaatatt ggtctgtgtt taagtttgtt atttgaatgt 109501 aatttacata gaggaatata ataatggaga gacttcaaat ggaaagacag aacattacaa 109561 gcctaatgtc tccataattt tataaaatga aatcttagtg tctaaatcct tgtactgatt 109621 actaaaatta acccactcct ccccaacaag gtcttataaa ccacagcact ttgttccaag 109681 ttcagagttt taaattgaga gcattaaaca tcaaagttat aatatctaaa acaatttatt 109741 tttcatcaat aactgtcaga ggtgatcttt attttctaaa tatttcaaac ttgaaaacag 109801 agtaaaaaag tgatagaaaa gttgccagtt tggggttaaa gcatttttaa agctgcatgt 109861 tccttgtaat caaagagatg tgtctgagat ctaatagagt aagttacatt tattttacaa 109921 agcaggataa aaatgtggct ataatacaca ctacctccct tcactacaga aagaactagg 109981 tggtgtctac tgctagggag attatatgaa ggccaaaata atgacttcag caagagtgac 110041 tgaactcact ctaaggcctt tgactgcaga ggcacctgtt agggaaaatc agatgtctca 110101 tataataagg tgatgtcgga aacacgcaaa acaaaacgaa aaaagatttc tcagtataca 110161 caactgaatg atgatactta caatttttag caggtagctt tttaatgttt acagaaattt 110221 taattttttt ctattttgaa atttgaggct tgtttacatt gcttagataa tttagaattt 110281 ttaactaatg tcaaaactac agtgtcaaac attctaggtt gtagttactt tcagagtaga 110341 tacagggttt tagatcatta cagtttaagt tttctgacca attaaaaaaa catagagaac 110401 aaaagcatat ttgaccaagc aacaagctta taattaattt ttattagttg attgattaat 110461 gatgtattgc cttttgccca tatataccct gtgtatctat acttggaagt gtttaaggtt 110521 gccattggtt gaaaacataa gtgtctctgg ccatcaaagt gatcttgttt acagcagtgc 110581 ttttgtgaaa caattattta tttgctgaaa gagctcttct gaactgtgtc cttttaattt 110641 ttgcttagaa tagaatggaa caagtttaaa tttcaaggaa atatgaaggc acttcctttt 110701 tttctaagaa ggaagttgct agatgattcc ttcatcacac ttacttaaag tactgagaag 110761 agtatctgta aataaaaggg ttccaacctt ttaaaaaaga aggaaaaaac tttttggtgc 110821 tccagtgtag ggctatcttt ttaaaaaatg tcaacaaagg gaaaataaac tatcagcttg 110881 gatggtcact tgaatagaag atggttatac acagtgttat tgttaaaatt tttttacctt 110941 ttggttggtt tgcatctttt ttccatattg ttaattttat accaaaatgt taaatatttg 111001 tattacttga attttgctct tgtatggcaa aataattagt gagtttaaaa aaaatctata 111061 gtttccaata aacaactgaa aaattatcat gagatgtgta tttaaacttt ttcatgaaca 111121 ttgcttatat aatcattcct tctgtcttaa tgtactacat ggtcttagcc ctgttcctat 111181 aggattatca tgttctctgc attatagagc cacctaagat gtactttttg ttaaatgact 111241 catgctggaa tatctggatg gggagatgtt ctttcctaat gtagtcatgt gccacaaaat 111301 gacgtttcgg ttaacgatgg atcacatata tgatgatagt cccatgaaat tgtaatggaa 111361 ctgccctata caggtgtacc attttttatc ttttatttca gatttttact gtaccttttg 111421 tatatttaga tgtgtttaga tacacaaata ctttccattg tcttacaatt gcctgcagta 111481 ttcagtacag caacatgctg tacaggtttc tagcccagga gcaataggct ctaccatata 111541 tctaggtgtc tagtaggcta ttccatctag gttctcgtat gtataacctg ggatgtttgc 111601 acatcgatgt ggtcacctaa agatgcattt attggccagg cgccatggct cacgcctgta 111661 atcccagcac tttgggaggc cgaggcaagt ggaccacctg aggttaggag tttgagacca 111721 gcctggccaa catggtgaaa ccccatctct actaaaaata caaaaatcag ccaggtgtgg 111781 tggcacacac cagtaatccc aactactcgg gaggctgagg caggagaatt gcttgaacct 111841 gggaaaggga ggttgcaatg acctgagatt gtgccactgc tctccaacct ggacgacaga 111901 gcgagactgt ctcaaaaaaa aaaaaaatgc atttctcaga acttatcctc attgttaagc 111961 aatgcatgac tataatctgt tgagagaggg atgaaatcac ctgtagttat agcgctttaa 112021 gataccattt gaaaaggtta cgttttcctt ttctttgaca cggttagctg tctgaaatac 112081 agtcaatttt aaccctaatc tcttaatatc aggaatgccc ttacacctac tttggagtgt 112141 ctggtgcttc gatatagttg catgtaatgt gctctcatct gtttttacct gattcctgct 112201 cagttcttca catggcatat tgtgtaactc aatctatatt taaaacttgt aagcatccga 112261 attatttgtt tatggtagaa ctttttactt gcaagtcgtg gtaggagtgt ttgtagttgg 112321 tactaaaatg tgatgacttg gaagaattaa ttaatgacct atatttgggg actttaattg 112381 gatgctatag ctgcaatgag aatagaacca gagaactctt gatatgcaag gttattcatt 112441 ctgtgataat aatgagagga atattcgatg tctctttgag tcagttttcc ttccgatcac 112501 ttccgcattc tgcagtgaca caatccttaa tcatagcttt cattacaata ttcctttact 112561 ccagacctca aagactcctc actgcctcca gcatcaaatc taaactcttc tacctggttt 112621 tcaacgccct acgtaaactt ttcctccttc attccctata gcttggtttt ttttttttta 112681 tcactgccac tattatctat cacaaatgtc aatcatgacc ataccttgct taagttggtt 112741 tcccttgcca agagcactgt ttttcctata cctgttgaaa ttttggaaat ccaattctac 112801 ctcctctctt cccttaagca tctcttcctt cccttctccc caaatttata tttagtcaca 112861 tatattatcg tactacctac taatcattcc atgtgttttt ttacgagctg taagttctga 112921 aggcaaccat gccttgtaca gtgtccactt agggttctga ataattaatc atctccccaa 112981 aatctgaaag ccttctatat accaagcaaa tttgtttagt tatgcagcaa aactcaaatc 113041 tataaaatca aaaaggaata aggaaataca gattaaacag ttgcagcaaa gactggtgat 113101 cttaaggtat ttagtcaaag ctggtggtag aacaaaaaca gtagtcttac agattctacc 113161 tcttgattaa ctcagtggct aattttgcct tttctcaaag ttcttttgca agaacataaa 113221 gatatttttg tttctttagt tgagtgctgt aactttattc ctttgtgttt ctcataagta 113281 tgatttggca gtctgccata cgttttttgt tttttttctt cctctttgag acagggtttt 113341 gctctgtcac ccaggctgca gtgcagctgt gtgatcacag cttagctcac tgcagactta 113401 gcttcctggg ctcaagcaat cttctcacct cagtctcctg agtaactgag actacaggtg 113461 caccccacca catcccgcta aatgttttaa tttcttgtgg agatggtgtc ttcactatgt 113521 tgctcaggct ggtcttcaac tcctgggctc gagcaatcct cctacctcag cctcccaaag 113581 tgttggaaag tgttgggatt acagacctga gccaccacac ctggcctgtt ataagttaat 113641 acaataattc atcaactaac ttaaagaaca ctaagactct tacaaaagta ggtatgagtt 113701 ttagtaaaag tctcaaaaga taaactgtca cttaaggaaa actagagaac atatcattgc 113761 caaatggtgt ttttcagaga ttataccatt caacgcccac atgctgaatt gggccattca 113821 ttataactcc aggaacatgg caatcagtaa gagcccacat gtttctttga atacaccgta 113881 agtgaaagaa tataaagtag tctagttaat attatgttta atcaaggagc acattcctaa 113941 agatgtttgt tcattcattc tacagacatt tttcagaggc ctgctaagag tcaagcatta 114001 tgatagacgc catggataaa agcttacaag tcaaccagcg tgggtataaa taatgtgact 114061 agcactagaa caggtatcat gatggggatt ctgagtataa atattttttt aaaataaatt 114121 tccccgtgtt atttttggct tttacctccc taatttaggc tttctaaatg gcacagcatt 114181 tctgaggatg caaacacctt tctacagagc aaaaacagca tttgtataaa tttgtgtctt 114241 tggggaacca agagacttta aatgtgttta aaccaataat tcagtcaata tcaacattag 114301 cttacatgta atattctctt gatagcccaa ttttttaaaa cactgtattc ttagaagttt 114361 ggtttctaag atgtcacttt aagctctttt gcttgttgct tttgtgggat ccacaaattt 114421 tgttctcagg tacataaatg aaggttagta tagaggataa atattatgat tcttatctgg 114481 gaaagacagg tgctgaggtg taaaagagag gatcctcgcc acccatgccc cgcaccccct 114541 cgccccctgc acccacggat gtgcagtctt acctgcgggg ggaaaggtct ccgagcctgg 114601 cctgctgctc cagctcaggg ttcccccttt catgatggct ttcaaagatt tcttcactct 114661 gaagtgaaag aaattttggt aagatttgat attgtaggga cctcctaatc tatatttttc 114721 tctctcccaa tttctgtgtt tgattttggt tttgagtctc tcgataagca aaatatccag 114781 tttctcatgc cgctttctca ggttttcccc agccactctg gatccattat ggtttgccct 114841 ttttggcttc ctttaggcac actaaaaact ccttcccaaa agcaggtatc ggccgggcgt 114901 ggtggcaggc gcctgtaatc ccagctactc tggaggctga ggcaggagaa ttgctcgaac 114961 ctgggaggcg gaggttgcag tgagccaaga tcacaccatt gcactccagc ctcagcaaca 115021 gagcgagatg ccatatccaa aaaaaaaaaa aaaagcaggt gtcctttccc cttaatcatg 115081 aagggctatt catactttac tgccccaccc ctattgattc ataagaggac agtaaagcga 115141 tcactgcatt caacatctcc tttttttttt tcttgtaaga aatcaaggtc tggaaaagtt 115201 gcactcgccc tgagaccaga aagtgctgga ggcagagatg aagtaagccc agtgtaggct 115261 ttcacagatg cgtgataaca agtctaacta aagaagtacc tgggatacta gttttgccaa 115321 ttcagctcta aaacaatatg gcaatcttat attccaaata tatatatata tatttgtatt 115381 tatagtagag atggggtttc accatgttgg ctaggctggt cttgaactcc tgacctcaaa 115441 tgatccactg acctcagcct cctaaagtgg aggaaacata tatatatgtt ttccttttaa 115501 aataggatgt cagtccaata agaatctaaa ttttagttcc ctctaatata tatatctgac 115561 tagggaccag ataatatttt tcatgtgtca atatataaaa gttggccagg tgcagtggct 115621 catgcctgta atcccagcac tttaggaggc tgaggtcagt ggatcatttg aggtcaggag 115681 ttcaagacca gcctagccaa catggtgaaa ccccatctct actaaaaata caaaaattag 115741 ccgggcatgg tggcaggcac ctgtaatcca gctatttggg aggctgaggc aggagaatca 115801 cttgagcctg ggaggcagag gttgcggtga gctgagattg caccactgca ctccagtctg 115861 ggcgacacag tgagaccctg tctcaaaaga aaaaatatat atatatatat attttttatt 115921 atattgttta ttaaacaaat aaaaataaaa gatatctcat cagttacgat gtaaatgaaa 115981 aaattgtgtg tgtgtgtgtg tgtgtgtgtt atgatgtact tcttgtgagt tgtggtcagg 116041 ctttgaaagc cactatgtat gtgtgtgtgt gtgtacataa aagtaagtat cagtcccagg 116101 attcaaattc gaaccagtcc cagtcaaatt caatctaatt attttcacca cactaccaaa 116161 agtctttctt cacattttct atataaagtt gaactaatta atacagcagt gaatgttaca 116221 ttgtatcctt ttgcagtttc tcatctatga cattactatg cctgaattcc ccactggact 116281 ttgaactcag tcttactcat ctttggatcc ccagcgtgta atacaggccc ttcataaaga 116341 gtgaccatta ttactaacaa aatttcctct cactgtgaaa cctgctccca attataaaat 116401 gaattgtgct ctttcgaact ccgttgatct attgtatgca gggtggatca tagagtctta 116461 tttcataata acctagtgat ggaaaaataa caatgattca ggagtaggta atggaccttg 116521 tcatctttta cactgaatga tgagatttcc ctaatttata gattttgtca tgcatgagat 116581 gccctcagga gcttgcagaa atgccttggc acgttgctct gctgacatgt gtcagatggc 116641 tggtgtggag gtagagggag tctcctctgc caagcaagtc taatggaata taacactggt 116701 gccattgagg atgcaatgag aaggacccac agcagatcca gagactgcat tcataaaagc 116761 tgcacagtat accatgtttt attaaggtat agacagatta gttgtgttct agcatagtgg 116821 ttttcaaagc ctgaccacaa ctcacaagaa gtacataaca cacacacaca cacacataca 116881 cacacacatt ttttcattta catcgtaact gatgagatat cttttatttt tatttgctta 116941 attttttatt tctttttttt ttgagacgaa aggtgaattg tagccaaagt acttttagga 117001 aattttaaaa ttacacattt tgaaagctct aggaggaaga gagcatattc aagtaaaacc 117061 tttcttttat tcaaatagta gactaaaaaa ttgaccatag actaactcag caattgctgc 117121 catatttctt agtgagggat tccttatgag ctccattgaa tgataaaagg atactcttcc 117181 aaatcagagc aacaatcgtt tcttgtgaca ctgcatcctt tttcccttcc ttcaccctga 117241 attgccccta gggtatacct aggagaggga gggctggcaa ccagctgctg ttgtgaaagg 117301 gatgtcattc atggcctgcc ctattgggag gccaatcagg cggaacagcc cctctcccct 117361 tatggtcatt ttatcctgaa gaaaaagggt ggcaaaaaga tggcaaaaaa gattgctgat 117421 cacagatcac tgttacaaat acaacaattg gctgggcatg gtggctcacg cctgtaatcc 117481 cagcacttcg ggaggccgag gcgagtggat cacgaggtca agagttcaag accagcctga 117541 ccaacatggt gaaaacccgt ctctactaaa aatacaaaaa ttagccgggc atagtggcgc 117601 atgtctgtag tcccagctac tcaggaggct gaggcaggaa agtcacttga acccggaggt 117661 ggaggttgag tgaaccaaga ttgtgccact gccctccagc ctgggcgaca gagcaagatt 117721 ccgtctccaa aaaaaaaaaa ggagggggtg gcaatgagac agggaactgc tctgggtcca 117781 actcccctca catagcccca acccccatat ccaccccaca aacctccacc tccccacttc 117841 cagcctctcc agtttcaaag tgacgcttac caacacgcag gctctccagg agcaccttga 117901 aaggtaactt cttacatctt tccaaaacac ttataatcta attgttcttt ctcccctata 117961 ttttgaagaa atgcatgctc tcaaaattga aaaaaacgcc tagaggaatt ttgtaaaaaa 118021 attatatttt tccagtttct ccaaaggaca tataaaccct ttttaaactt agattttgaa 118081 aagtcccaat agctgatttt caatcgatta ttgaaattgt tttttttttc ttccctaaag 118141 gggagatacc aacctttgaa caaaattaaa ttaacttttc cccaaagtta aatgatttta 118201 ctttgtgatt ttaggaatgt gtagaaagtg atttcagttc caactctttt aaagcaaggg 118261 tatctgggcc aggcgtggtg gctccctcct gtaatcctag cgctttggga ggccaaggtg 118321 gggcagatca cctgtggtca ggagttcgag atcagcctgg ccaacataga gaaaccccat 118381 ctctactaaa aacacaaaat tagctgggta tagtggtgcg cgcctgtact cccagctact 118441 cgggaggctg aggcaggaga atcgcttgaa cccgggagac ggaggttgca atgagctgag 118501 atcacgccac tgcactccag cctgggcaac agagtgagtc tctgtcaaaa aaaaaaaaaa 118561 aaacaaacaa gcaaacaaac aaacaaaaac aagggtatct ggtaatttaa ggtgaaagta 118621 ttattcacta ttataatcaa ttttcttaaa aacaggagct attaggccca attctgagga 118681 gagaaaaaaa agcagatttc aagggcccgt gccaccttct ttgtttaaag catagagaat 118741 gataataaaa tgttaaaggt tcagcatttt ctggcattgt aaatttaatt aattaattat 118801 gtaaattaat tatggaaatg agaaataaag tgaaaaggta tgtcaagtaa gaatcaataa 118861 ttatcaaaac gttctctacc aaatgggatg caggggagaa gggtgagaag aagggaagga 118921 aataaaggca aaggcatttg cccacagcct cagtccaggg tgttctacat gacgcagccc 118981 cagagtaaaa aatactagcc acaggaatga gtggctctgt cttcctctcc agaactgacg 119041 ggcaaaagag ggggaacctc accttttttt tttttttttt tttggaaact cactgggttt 119101 tgatatggag aacaggaaga gaatctaccc tagcagcaca ttcacagatg gtttgtctgc 119161 cctatttgct ccatctccct ggctcttcct agcacttctt ttcctacttt ccattttcat 119221 tggccgcaaa agccacttaa atattccttt ggaaaactgg gcagtctgtc ccctttttaa 119281 aaccacaact atttccagga gagttaaagg gctttcggac tgcccatcat tcatgcattg 119341 tggtgaacta gctcagactg cctctacctt tagtccagat cagctgcctt gggagggcga 119401 ttcctggagg aaaggatgag gagaggaagg ttttaggaca tcgtatggac tgaagccccg 119461 ttggagggga agggcaggct tgaggaacaa gcctcagtgt tcctgaggct ttgtggaaat 119521 gagaaatgaa tagagaaaga taagagactg gatcctaaat gcaattttgc ctatttcaaa 119581 attttcacag ggtctttttt tttttttttt tttgagacaa ggtcttgctc tgtcacccag 119641 gctgaagggc agtggtgtga tcatagcttc ctgaagcttg cagcctcgaa ctcctgagct 119701 caagcgatca tcctgcttca gcctcccaaa gcgctgggat tacaggcatg agccaccacc 119761 cccagcctca gggtcttttt aatccctaag atcttaaatt tcccagctac tattgttagt 119821 aatgagttca atttacttac tggttaaatt ggcttttatt attattatta tttattttat 119881 tttattttat tttatttgag acagagtctt gtgctgtcgc ccaggctgtg gtacagtggt 119941 gcaatctcag ctcactgcaa gctctgcctc cctggttcat gccattctcc tgcctcagcc 120001 tcccgagtag ctgggactac aggcacccgc caccacgccc agctaatttt ttgtattttt 120061 agtagagacg gggtttcacc atgttagcca ggatggtctc gatctcctga cctcatcgtc 120121 tggactaata ctctaaacgc tattggcaat agtttgttta caggaaaaac atcttcttat 120181 aaagctgact gcaaatgttt taataaattt gcaaatatgt aattctgttt aaaatatcag 120241 gaagtgagaa acatgttatg tgataactct ttcccattcc cgaaccaaga aaatgtaaag 120301 gcagtaagtg tgccaaggac actgaaagga agctgcccgt acatctttga atcttctcag 120361 gctgtttggt ttcatctgat tttaagctcc atggataaat ttcattgtaa caatttttat 120421 gtctgtaaat cttaacccat aaataaaatc acaaatcaga aaagtacttt attaggccag 120481 tctttgtcca agcaaatttg gtcccaaagc tagtttacaa ataattgcca cacagctaca 120541 aggccagtgg gttgactatc gtagcatcaa tggtgtgggc agggctgcag cctctggggc 120601 agcattagcc ccaactgaca gagggtatag gtgctcttaa caatctatga gaccccccac 120661 aacctggact cagctgtcat aagcctttac ttcttatgtt cttcctaagt actttatcgg 120721 gccaacaaat tcatcccatg aggaatccaa gatggaaacg tcaaaactat ctttggtatc 120781 atgcttcctc cccctacctt ctctgccttg atcccctcct tcattctaca cattttctct 120841 gatagtcgtg ttctagccac tagggagaac gtttcagtca atgggtatga tttctgctca 120901 cctctttttg ccatagctat tagtaagctc tcaccactga tgtctcagct gactgtgaca 120961 actgccagcg gctggtcagc ctgcctgcct tcatctaacc tgctctttac tgtctaccag 121021 agtcagcttc gtaatacaca ggtgccctcc tcctcctgtc aacattcctc aaccaaagcc 121081 tcagtaaact gtctctcatc taccttccca gcattcttga gccattccca ggatagccta 121141 ggccctttag acctctgaga tgttataccc ttcctcattc tggaatgccc ttcagtgtct 121201 tacccacctt tcagagttct atttattctt tttttttttt tagatggagt ctcactctgt 121261 cacccaggct ggagagcagt ggcaccatct tggctcactg caccctctgc ctcccaggtt 121321 caagtgattc tcctgcctca gcctcctgag tagctgggat tacaggtgcc cgccaccatg 121381 cccagctaat ttttgtattt ttagtagaga cgaggtttca ccatgttggt cagtctggtc 121441 ttgaactcct gaactcaggt gatccaccca cctcagcctc ccaaagtgct gggattacag 121501 gtgtgagcca ccacgaccag ccttatttat tctttagagt tgaattcaaa tgccacctcc 121561 tttgaaagcc atccttgatg tccttgtgag gaattacttt ctctaccaca ttatatattc 121621 cttttactgt agaaacgtca ttctgcttta tattataaat tattagaaat atgttgtttc 121681 ccccatgaaa gtttcaagtt tcctgagttt aggcctgatg atacaatcat tttcatatga 121741 ctcacagcaa ttgccttagt atatattaaa ttgagctgaa taagccatgt acccagttga 121801 acacaccaaa tattttagtg atgtcatttc tttattggtg aaagagcaag gccaacgttg 121861 atcttaatgt tattctctct tttatcttat tcagctccct caacaaaaat acattctcat 121921 agattaatgt aatcaccacc tcactcctct caactttaga ccctatacta gcctgagaaa 121981 tccagatcca acttaatgag ttctccttta tcccaagtcc tcatgaccta taaatgtaac 122041 ctctaggaca atgttacaaa gagtgagatc tttataccac ctgcattctt gtcaccaaac 122101 gtgtaagtta aaaattcaga ttctgagtgt ggtgactcac gcctgtaatc ccagcacttt 122161 gggaggctga gccaggtggg tcacctgagg tcaggagttc gagaccagcc tgggcaacat 122221 ggtgaaaccc tgtctctact agaaatacaa aaattagcca ggcatggtga caggcacctg 122281 taatcccagc tactctagag gctaaggcag gagaatcact tgaacctggg aggcggaagt 122341 tgcagtgagc tgagatcact ccatggcact ccagcttggg caacaaagtg agactctgtc 122401 tccaaaaaaa aaaaaaaatt tagattccag gacccgaccc tacacctccc taattagaat 122461 ctcagagagt ggggccctgg gaatctgcat aattcataac cttctcagag ctctggtaca 122521 caagaaacca tgagaatctt tgttctggaa acttcagaga acttggctga gggccacccc 122581 aggatttggt ggcgtccctg aatctcccat tagctctgac actgactaga cttcaaatat 122641 cacagaaggc aagcattgaa gtgtgtttat attcaaatag ttttcttgtt gagaatcaga 122701 aatattaata aaacttttgg gagtcaaagt aatgagaaga caaggaactg aacttgcccc 122761 ctgatctgtt atcatttggc agtaaggtac atattcaaca ataggataat tctgtataag 122821 acctgaatct gaccttcttt catctttcac actgacaatt ctgtctctaa acacaggatt 122881 aaatgaggaa ctaaaattca ccaagatcct gctaggtgtc aggtacagtg ctagctgctt 122941 ccacacattt ctcatttaat cagccacctg tgaaattgct atgatttatc accatttggc 123001 agaaaaggac atttagggca caagagatta aataagctgc tggttgggta cattaagtat 123061 taataaataa tcagatagat aagttaggta gttaagtaag ctgccagatt agtaaacagc 123121 tagtttcaaa tcccagctcc aacatttact agttgtatga cccttgggga aactcaacct 123181 ctgagtctca tggcactgct gataaaacgg aaaacatcat cctcacaagg ttctgaggac 123241 tagggatgga ctcaggtaca cacagcaccc agtacaatgt ctggccttca gccagtgata 123301 gcaacccttc actcacctct ccattccctt ccagcccctg cattcaaaaa cttttattta 123361 tttatttttt taatttagga ggctctttgc tcagaatcct gaaaaggctt ttgttacttt 123421 attttttctt ttttttcatc ttctgggagc acagaatagg cttctgctcc tttaaataac 123481 tttaacaggc accaaaggaa cactgctagc tctttcttaa ttctgtaggt ccacatttag 123541 gaaaaagaaa ttgtcagcct ctgacttatt tccagcttac aaaaaggctg tgatgttggc 123601 agcccgggga aagcagttcc cgtgagtcat gctttcacct ttcagccagt gaagaacagg 123661 aaatagtcac atcactattg ccaacaagca cagcgaatcg cttccaccac cgggctttcc 123721 cagatgacgt cagtgagcca agtgcagggc atcacccttg ccagatggcc ccggaagagt 123781 ctcagctgcc ctgtcaagtt tagcttctca agctccccag aaccagcatg gcaaggatca 123841 cccctccaga aaaggaaatg atttctctga tcatgatcaa accatgtcat aattttagct 123901 gtaggtggtg agtatcagtg actgtattat agatggtttt ggttcacagc tatttttttt 123961 ctcggtatat ttactctcaa gggcaaaggg gtggcattta tcacaatgct atgattcact 124021 ctactagact ggctgggttt catttcatct ttgagttcta gacctaaggc caaaaggatg 124081 tcagaatcgc cacccagagc tagatgcatg ctcaatgcaa cctggccatc tctctcggac 124141 agtggccact aaacacaagc tgcaatgatt attgttgatg ttagtcaatt gatttgtccc 124201 ccttttaata atccatgatc cacacagaca tgaatctaaa cctttttttt gaacctatct 124261 ttttcttact agttttctta catattttgg aataacaatt tccctggtta taatacacaa 124321 tgtgtgaaac actaattgca tttattggtc ctaaattcat tcccaagtct aggatttggg 124381 gatttagtgc acaaatccac tttcttcctg ctcatgttgt tcatgacttt atggattctg 124441 cttacatttc ctcccaaagc cttggtcttt ctaagcagaa atctgaccag cctctgttcc 124501 tactctgctt cctccaccac cttgactgtt aacatacact gttgtgggag aagcttttca 124561 ttaaatacgc aaacaaatca acaataagga agaaaacaac tgaagcacag aaggatgatt 124621 gtaacatgga cttgtgcttg tttaacagta cccatactgt ttggtgagtt gcctaaatac 124681 agagggagat ggcaacttaa caggatgggt ttggagtctg aattaaataa acaacatggc 124741 aaaagaagga agaaaccaaa agttgggtga ctagggcttc ttggattcta ctcaacactt 124801 tccacaccta caggcctctg attctatccc caccgctgct cctacactgt ctatgtttct 124861 cttttacaac aacaacaaaa aaatagcact tgtttactgt tcacaattat tgaaataaat 124921 cactctaatt tggattcctt tatatgagaa cttccatatg cttaaaactc ccgtagcatt 124981 tttatagtgt acaggtcata aacatgttgt ctgaagatgg gcaaagtagg tcgtagagat 125041 aggaagtgag ttcacatcac ggcgtgagtc aaggcagcag catatgagct caggggccaa 125101 atctgagctg tgatcatgtg acacagtcct caccttcaga gatgacattc tcccagaagt 125161 ccagggtgca gaaatggata ggctgatgtt cacttttgaa gaggaccaca atgaaaccct 125221 tttaatatta ctcaatgatt ccacagacct cctgggtgtt aaatcaggtg aatctatttt 125281 taaaaattgc tatttacttt tttaggtggc tgatcaggac agtatgaaat ttcatacagt 125341 ttcctaactt gagaaaacat ggtgatgcaa ttcctccaac caggagggat ttatggacag 125401 cggtggctac gtcctaatct gcccagaata caggatgact aaacaaattg gcaaacggac 125461 gcagctttct ctctctctaa gaaaagtctg ctgagaaccc tcgttcccac tctgtttctg 125521 cctccaagaa gaaaagccta aaactcactt tcttccggac tctttcaagg tcagtggagt 125581 tcctctgggg ttcatattca gatactttgg ggattgatga tggatcataa atgttgctga 125641 agttcatttg agcccatggc ctctggtttc agaaccattc agccagtcaa atatttaaat 125701 tttagtgagg caggggaaag ggaacccctt cgggggctgc tatacagcaa cttatattca 125761 caattcacaa ttaaatacac ttcagtttct aaatatatgt tactttaaaa ttatacagtg 125821 cttagcaatt ttcaaatttt cattttgtgc tcaaaatatt ttcaggaggt agctgttgtt 125881 atacccattt gtacaagcaa gaaactgagg tttcaagagg ttatgttgct taaacccaga 125941 tctgcctgat tccaaacctt atgctttttt taaacttcta ttttgaaata attatattaa 126001 gtcacatgag gttgcaaaga aatggaaagt ctcatgtccc ctttctccaa acctcatcca 126061 atgttaacat ctaagaaatt gacactggca caatccacag agcctattca gggttcacca 126121 gttattcacg cacttgtgtg tgtgcatgtg tgtgtgcagc tctgtgcaat tttgtcatgc 126181 tctttttttt tttttttttt gagacatagt ttcgctctgt tgcccaggct ggagtacagt 126241 ggcacagtct ctgctcactg taacctccaa cttccagttt caagcaattt tcctgcctca 126301 gcctcctgag tagctgggat tacaggcatc tgccaccacg cccatctaat ttttgtattt 126361 ttagtagaga caaggtttca ccatgctagc cagactggtc tcaaactcct gacctcgtga 126421 tcttcccacc tcggcctccc aaagtgctgg gattacaggc gtgaaccacc gcgcccggcc 126481 tgtcatgctc tttttataca tgaaccatac actgttctgc caattttaaa tatgaaggca 126541 gattacagaa ataaataaaa tgatttttct tttactacaa cagtatctac caataatcac 126601 atacatgacc aaatagcttt cacttctagc tgccgttaag aagaaagaaa aggatgaaaa 126661 gaaaaaaaat cctataaacc ccagttcttg aaagcattag tctgtgctat gtgccttagg 126721 tacagattaa ggaaacacaa tttgttgatt tattgataat tgtgacagca atcttccctc 126781 ttgtcaggaa gttctataag taaaataaag gtaattttac cttgacttca Ratttagtct 126841 catcatcttc ctttccccgg gagttcaact ctgtctccgt ataaggttcc tcagacgttc 126901 tcagaggacg agctagaggg actaagagaa cctgccatta gtgtggttat ttaacttata 126961 aactataact cagtgcactt tgtctgattt ggacaaatag ctggaccacg tcaatgtggc 127021 taactataaa agctcacttg agctgctgcg ttgatttcct agggctggat gaaattggag 127081 gtgaaggaga gacacagagg agaaatagaa tccttgactc cagggacttg acatttcagc 127141 agaagagaaa aaagtcacac ccagcaccat atgaaacatt aaactacaac aaatctgtac 127201 atatagcaat gtggtaaaga atagtagatt ttctgtcatt cccattttta aggcccagct 127261 aaaaggtcac ttttggctgg gtttggtggc tcacacctgt aatcccagca ctttgggagg 127321 ctgaggcggg cagatcacct gaggtcagga gttcgagacc agcccggcca acatggtgaa 127381 atcccgtctc tattaaaaat acaaaaatta tccgggaaca gtggcaagtg cctgtaatcc 127441 tagttactcg ggaggctgag gcaggagaat cacttgaacc tgggaggcgg aggttgcggt 127501 gagctgagat tgcgccactg cactccagcc tgggcaacag agctagactc catctcaaac 127561 aaacaaacag acaaacaaac aaaaaggcca ctttcctcat gaagtcttcc atattacaaa 127621 ctttatggat tcttttatgc actgctgctt tggcatgcat catgtgtaca gcataatgtt 127681 gcacttgatg ttgttcttgg attcaaagac taaattctag gtactacatt gtattgaata 127741 tattttccat tataaaggta attaattttt gcttattaca gaaaatctga aaaacactaa 127801 actcttaacg ctgaagcaca atcacttagt atatttcctt acagtcttct ttcaaatgct 127861 ctccctcttt ttacacacat acatgtacac acacaatcat actatgattg tattgtaaga 127921 ggatggccat gcctgctgct cttgctcttt tctcttggcc actctatctc ccttctcccc 127981 acctgggaaa tatcctttct tcaagctcca tggccatttg gtagtaaaat tgggacttga 128041 gaggagaagg cctcaaggct tcctaaccca ccttaccagc tttgccaagc attgtgggtg 128101 atggccacaa ggctaataga taagaggtac tttgaattct tatttgtcac agtcaccacc 128161 cttgcatatg ctggtccctt gggaaactca caggagacat gattaatccc aggcaggatt 128221 tgagcatttc tttctttctt tctttttttt tttgagacag agtctcgctc tgtcgccagg 128281 ctggagtgca gtggtgtgat ctcagctcac tgcaacctcc gcctcccagg ttcaagcgat 128341 tcttctgcct cagcctccca agtagctggg actacaggcg cgcaccacca tacgcagcta 128401 atttttgtat ttttagtagc gacggagttt taccacgttg gacaggatgg tcttactctc 128461 ttgacctcat gatctgcctg cctcggcctc ccaaagtgct gggattacag gcgtgagcca 128521 ctgtgcccag ccaattttag catttgtagg tcccaagacc aaaatgccat tcattgaatg 128581 ccaccaatat atcaccctga ttcttagcta tggtagactt gatggaggtt gaccatctga 128641 tctccagagg ccccttccaa cccttccatt ctgtgagcca actggaagca gcttgggctt 128701 tctatgggtt tttcatagat gttatgttga aaatcgcaga aaattacact gctaccccag 128761 gttatacata ttaccattaa tgctgctttg taataacaga caatcctttg ggctcctccc 128821 tctctgtggg atctctataa agtgagtgat tccaggcaca ataactagat gctagaaatg 128881 catgcactat aatttgtgac accatgactg agaagcgcct gtccttgaca ctgtaagtag 128941 agtgaagcag tgaaaggcgg ggccacagaa gccacactgc cagtgttcaa accttgctct 129001 tccatgcacg agacaaacat gattacagtc acctctgtac cttacctttt tcatctgtaa 129061 agtagggaaa aatgagaaat cctacagtaa agggctgtga taaggcttta atgagttcat 129121 ttgtgaagaa tgtagatcat atacaatgca taacacattt tagctattat tgccacatgg 129181 cactaaaaag ttttttcacg ttccttatga acttcagagg tgttagaagt tgtagccaag 129241 gcatccagct aacaagagtc agagctgaaa ttccagctct taagcccctc cacacatctt 129301 ctttatactg tgaaatacaa ttgcttatct tggcacagac tcagttacta aggaatcaga 129361 caaaaatgtt tgaaaatcct tttagtgact tgctggagtc cacactcagt agatacttac 129421 caaaaacaaa agtgcaggat tgttctgcaa aacgagctag ttgtttgact taggtcaatg 129481 acaacctgtt cagacttgac aaagacctat ttattctggg aaggagtgtt atctattatg 129541 aaatcttgta tttgaaaagc tgagaaacaa ccctgtaatt cctgctttat ttacttgaaa 129601 caaacatatg tacctggaaa caacgaatga ggccaagtca tagatgtgaa catagtttag 129661 cttgaggaca gacgaaaggt caagagaacc aagttctgtt tccaatcatc taagaaggat 129721 gtctctgtgt atgtttgggt atgtctgtgg tatgtgtgtg caagtgtgtg agagtgtatg 129781 tgtgtggatg tgtgtgtgtg tgtttgcaga taaaaattca ggaaagaaat acactaaatg 129841 ttatcaaggc ttacttctaa aaagtaggat acatggtgga agacaaggga tcgtaaattg 129901 tactaaatgg agaattacca aaacacccca caattttcat ataaacaaaa actgaaagaa 129961 tttattgcta acaggccagg catggtggct cacaactgta attccagctc ttagggaggc 130021 agaggcagga ggatagcttg agcccaggag ttcgaaacct gcctggacaa tatagcaaga 130081 ccccgttctc cacaaaaagg aaaaaataaa gacaagaaaa gaatttgttg ctagtagact 130141 ccccaacaat aaatactaac agaagctctt tattccgaag agaaatgaca caagatagta 130201 attcaaatct ataataagga atgaagaacc ccagaaatgg tgaataaggg ggtatgtaca 130261 cacacacaca cacacacaca cacacacaca tgcacacaca cacgtatgta tttctcttaa 130321 tttgatacat gactatttaa agcaaaaatt ataataccat agtgggggat taacatatat 130381 agatgtgata tttatgataa gaatagcaca gaggatggat ggggagcata tggaagtatg 130441 acattgtagt atgtagtatg atttcctata ttttgcatag aatggtatga aactaattca 130501 atagatcatg acaagttaag gatgcatatg gtaatcccca gtctggtgaa aaacagtaca 130561 aagagatatg ataacaagcc aatagaggaa ttaaaacaaa atattaaaaa atatttacta 130621 acccaaagga aggcaggaaa ggaggaaaag aggaataaaa agcacatgag acatgtagga 130681 taacaaaaca ggaactctaa atccaactat atcaataatg atgatggatg taaacataaa 130741 aatctaatca ctccaattac aaggcagagg tcacagtgta taaaaaggca agacccaact 130801 atatgctgcc tacaacaaat atactttaaa tacaaagaca gaagaataga aaacaaaaaa 130861 aatatgttct gcaaacacta agcatgagaa aactggaatg gctgtgacaa tatcgcacaa 130921 gatagacttt aagagtattt ctagaggtaa ggagggaagg agagacattt cataattata 130981 aaagagctaa tatatctgaa agacataaaa accatgaata tgtatgtgct taatgacaga 131041 ggtccacaat acacgacaca aacaaatgac atgtacatcc tgcccaaggg cagtttattc 131101 caaggatgta cagttgtttt aacatttgaa aacaaatcat tgtaatttac catattaaca 131161 aataaagaag aaaaaccata tcattctctc aatacatgga ccaaataagc atttgaaaac 131221 tcaataatca ttcacgataa aaactctcct aaaaaataga aatagaagag aattccctca 131281 atctgataaa agacatctgt ggaaaaccta tagcttacat catacttaaa gatgaaactt 131341 gaactctttt cctaatattg ggaaaacaca cagatgtctg ctctcaccat ttctatttga 131401 cattgtagtg gaggtcccag tcattataat atgacaagaa aaaaagtata aagattgaaa 131461 cagaaaaaag taaaagcatc cctattcaca gatgatagga ttatatttct atatagaaat 131521 ctattcctat tcttaattat atatggtaaa ttccattcat tatagggtta aaaaaatgtt 131581 aaatgcctaa ggcctttaca ctcaaaacga ctgcattgtt gagagaaatt aaagatgacc 131641 taaaaaacat aagttataac atattcagga attggaaaag tcaatattgg taagataata 131701 gttctctcca aattagctca tatatccagt gtaatcccta tcataatccc agcagaaatt 131761 gaaaggtgat tctaaaattt atggaacaat gaaaaggacc taaaatagcc aaaacaacct 131821 tgaaaaagaa caacgttgga acatcgcatg acttgatctt aaaggctgac taataagcta 131881 tggtaatcag gaccatgtgg gattggcata agaacttacc tattttttaa atctttgctc 131941 aaagaaccaa ttttactact ccattgctaa taaaacatgg gccttttaaa ggtcctgaat 132001 ggggtctatt ttatgctgtt attattatgt tattattatg atgcatttgt tgttgttgtt 132061 gttgttgttg agacagagtt ttgctcttgt cgcccaggct ggagggcaat ggcgcaatct 132121 ctgctcactg caacctctgc ctcccgggtt caagcaattc tcctgcctca gcctcctgag 132181 tagctgggat tacaggcgcc cgccaccatg cccaggctaa tttatttata tatatatatg 132241 tatatatatg tatgtgtgtg tgtgtgtgtg tatatatatg tgtatataca tatatgtgta 132301 tgtgtgtgtg tgtgtgtata tatatatata tatatttttt tttttttgag acggagtctc 132361 gctctgtcac ccaggatgga gtgcagtggc gccatctccg ctaactgcaa gctctgcctc 132421 ccaggttcac gccattctcc tgcctcagcc tcccgagtag ctgggactac aggtgcccac 132481 caccacgcct ggctaatttt ttgtattttt agtatagatg gggtttcacc gtgttagcca 132541 ggatggtctc gatctcctga cctcgtgatc tgcccgcctc aggctcccaa agtgctggga 132601 ttacaggcgt gagccaccgc gcccagccta atttttctat ttttagtaga gatgggtttc 132661 aacatgttgg ccaggctggt ctcgaattcc tgacctcaga taatccaccc gccacggcct 132721 cccaaagtgc tgggattaca ggcgtgagct accgcacccg gcctttatga tgcattttta 132781 ttattccttc agtagattgt gtgtttctcc ttgcatccag gcacttggtg ttatctaagt 132841 gtttatgtct tccatgtctg cactatgcta tatctgtagt ttgttttaaa tagtatctgt 132901 taaaacagcg accattatcc attgtagaca accagtcaca gtcctcatgg tgctgatgaa 132961 acacaaaagc cacaaagctt cttttcctca aataacctac ccgaggtgga gggctggctg 133021 gtttttgatg acacccattt tggtgaagac tcaggatgtg gggccacagg ttgcactgga 133081 cgagtctgtt tggctgccag tttcatcaga ctcacttgcc tcttgtgaaa tatttcctgg 133141 acatcatcca gcctctgcaa aactttctgg gctttggcct acagtaacaa aagcaaatca 133201 tgatgaacag gtctctctgt atacagcctg aggacacgaa gcattccctt tctcccacct 133261 tcccctactc cccccatccc tacctccctc aggtgacccc ctctgggcat cactatgcaa 133321 gtcgctgcag gtcctgccat gctccagaat tgggtatcta aggattagcc tctcctactt 133381 gagacccttg aggacaatga ctacatcctt tcactatggc gtctccagtg cctggaacac 133441 cctggcacag gagtttgaga ccagcctggt caacatggtg aaatcacatc tctactaaaa 133501 atacaaaaat tagccgggtg tggtggctgg cacctgtcat cccagctact tgagaagctg 133561 aggcaggaga atcgcttgaa cccaggaggc agaggttgca ctgagccgag atcatgccac 133621 tgcactccag cctgggcgac agagcgactc catctcagaa ataaacaaac aaacaaacaa 133681 ttagaataca atgtggcttg tgtcatgtta gacagagcaa agaattttgg cctgagtcca 133741 tggatggact tcagggcatt ccccagaatt acacaaacag ttagacaggc tggatataat 133801 ctgtttgttc ctccactgca tcttctcttc ctttctttgt gcccaggaag ctgattaaaa 133861 gaagctccta aaccagaggt gcagacaagg tgatgatgtg gctctcagag gaaggtgtgc 133921 tacctacaac cctgctggct tcatggaaga atatgcttct caaatgcaaa tccaatcaag 133981 ttatctccct gcttaaatcc aaactcgctc atgtggccca caaagcctac cttgcctgcc 134041 tctctctaac ctcacccaaa acacacttcc attgctctct aggttccagc accctgacct 134101 tttggtctgc actgtgccag gctccctcca gccctgaagc ctttgcacat gctgttcctc 134161 ctgtctggaa aggctctccc atcttgtcta ttcgttctcc aggctcaagt gactcttcct 134221 gagagccttc tctgacccca gtccaggtca agttcctctg tcacatgcct gaccctgcat 134281 ccctccttga cagcacttat atcagcttgt aacacatttt tgtgtgatta ttttgttaat 134341 gtcagcctct cccacaaact gtaagctcag tgagggatgg aagcatgcct atttttaatc 134401 atcatggtat cctcagcact cagcacagca catggtacat caggaatgct cataagcatt 134461 aatagagaaa tgactgattt gagtcagacg aagactcagt atgccaggac gttcaggccg 134521 agagtctaag aggccgccgg agggcttcgg aaccagtgag cccactcaat tcagcctgct 134581 cagtaccctg tgtgtacttt atcaggaaga aggtacagtc cctccctccc ctatccgttt 134641 cccaaccaca ggatcaaaat aacccggaag cattaccttt gcatcgaggg tgagcagcaa 134701 ctcaaactcg ttgtaaaact ccttggggct gagcaacggg tactccttga ctgtgcccag 134761 gaatgtcgca atgtcgttca aggcgatatc aaccccttct cgagactggc acttgtctac 134821 agcttgggaa gccaagaggt agattcctgc ctcacaccat tggctgacct tttggaaaga 134881 aacagtgccc tgtgcacttc gcctgaccac aactcagagc caccgtaatc ctcagcaggt 134941 ctgagcttgt aaggtgtcta caaggattcc cagacaatgg gaaattgtca tgggacggca 135001 tcgagtttaa ggggtctaac cactgtaaat tacactatga gggggacaaa cccaaaccag 135061 ctgatacctg tccacttctc agggaagtca ctcggtcagc aaaatgagtc tcttccatta 135121 acagtaattg ctacatctcc aggaggagac agctaaatat atatttaatt aaagacaggg 135181 tttcactctg tcacccaggc tggagtacag tagtgcaatt atggcttaat gcagcctgga 135241 gaggtctacc ccaggctcag gtgattcttc caccttagcc tcctgagtag ctgggactac 135301 agatgcatgc caccacacca ggctaatttt tccttccttc ctcccttcct tccttccttc 135361 cttccttcct ctttctttct ctctgtctct ctttctttct ttcctttttt tgagacaggt 135421 tttggccatg ttgccgaggc tgctctcgaa ctcctgggct caagcaatcc tcccacctcg 135481 gcctcccaaa gtgctgggat tacaggtgtg aggcactgca cccacccagc cttatagcta 135541 aatatctctt aggctggtct aaccatagag aaatgtaatc aatggctttt ggggtttacc 135601 tgctagtcca ctcttcttgc atccgaattg gttaccagaa cacagggtga ttaataagac 135661 attagacctg gcccttacat ttcctgggag aagggcatgt cccttttaac aaaaacacaa 135721 ctttcatctt tggattccca ggtgatccct atttgcattt actggcgttt gtctttcgta 135781 aggaagcaag aatgcaaagc tttgtaactc agcacagcga tactcaagag tttctggatg 135841 ctgggaggac attagatagg tgatgagttc taattattca atttttaaaa aagactcatt 135901 ttctttgctc ttttgaactc cactgagaca cttgaacaga aagacaggga gcccttatgc 135961 tacctgaaga gttatatgaa aagaagtttc cagtgccaat acctctgcat ttccatttac 136021 catgtgacct gaaaagcaat ataaagtaac tatacagaga aaaaagatca aaatgcaaca 136081 cgccaaagtg gttgtctttc taagtgggaa aatgataggc aacttaagtt tttgtcttat 136141 ttttttccta gattatttac agtgaatatg taccacattt agaataataa aaaatcataa 136201 agatatttca gaattaataa ttaccattat gtagggatgg gggatagtgg gatatgtacg 136261 tgcccatgag taaccttttt cttcctacat ttttggattt ccacaataat atgaactcat 136321 gctttgatat atgctgtgaa aactttctct ttacacatca tgcccagatc tgcaaaaaac 136381 attaggctgg gattttctgt agagttacta tggctacttg ctttctctct ctctctgtct 136441 ccctctctct ctctcaaaca cacatgcaca cacacacaca cacacacaca cacaccaggc 136501 ttataatctc tggagcaaaa atggagttgg gaaaacagca gctgatccca cagcgagctc 136561 agcacacaga gcaggctgca caccagcccc ttcggagcta cttgcttctg aaacaactat 136621 atttcattga cattaaaatc cctttagcaa tgggatgagg taagcgcctg ggtgggtgcc 136681 ccctgccatg caagaggaac agctgtgggg atggagcatg ctgactcatt gccgtcagcc 136741 acatgctgcc tgggaccagc tcacagggaa gactccagaa accgctgctc ctgctgactg 136801 ggaacatacc ctagaggtca cctccaaagg gcacccctct ccttgtgggc ccctggcatg 136861 cgcccttgct gtctatcctg tgaagccttg gtgtggaatc tggcaggcct gactttaatt 136921 ctggcttccc ctgtgtgcac catgtgtggt cttgggcagt catttacccc agggtttctt 136981 ggtgacatcc ctgcaccacc tgcaccagaa tcatttagac actttactaa aatactagtt 137041 cctgggcagt attccagacc tatcgacgct gagtcttcag ggcagtgcat aagggaatct 137101 gccatataac agctcactag gtgattctcg tgtgctacat tttgagaccc agcagagtat 137161 cagtttctgc ctctgcacaa tgaggccatt aataccttcc tcagaggttg tcttgaggat 137221 tataaataga tgacataatt atgttcctgg taggaatgca gtaggtgctc gatacatggt 137281 agggggtata aaatggttgt tattacttaa gcttctccaa aaagagtcaa aaccctttgc 137341 ctttatatga gggaagcact ctgcaaatat agtgcttggt tctctgtaca cctgaaaggg 137401 aatcaagaag tgttcccaca gggccaagcc aaaacacact cttaaagtcc ggctccacac 137461 agatagaaat gggcttcagt taactttaat gggatgtggt gggatggcca gagcactttt 137521 taccttcgtg ccaggtatac ttctacaccc caggatctgc acagttttta atgcctctcg 137581 aacatctttt cacataaagc actcaagcct ctcagcaatc acacaaggaa gggaggacga 137641 gtatgatgga agcttcttca cagaaaatct tgcagacatc atgttcttgg ctgtgttttg 137701 ctcaccttgt ccagctgtct atgaaactct aaggactttc ctaaaatgtc ccattttttc 137761 ttgtttccat tgatgaaatc gtcacagagg tgcctgagct ccacacaccg gggcctgatg 137821 gcatctgctg cataatggtg gctttggatg agctggtccc caaccagtgc cagcagcWgg 137881 gccttttcca ggggctcctg caaagtgaac acccacagcc aggcgtcaga agtcagagca 137941 tcatgaggct gagagctgcc tggtactgtg tgctccaagc ctggagggag gctgtggaga 138001 tgctaaccca ggatgaaggc tggagaacct gagaaagctc ggaatggttc caggcccagg 138061 aagttaagcc tggcccagag ttgtccacat gctgaacaac agcagtcatt cattcaaata 138121 cctgttgagt tctgggctag acatcgagga tatagtgtta acaagcccaa aattttcaga 138181 ttaatggaga attcagatat caactaaatt acacaaatcg ttaaattatt acaattgcaa 138241 taaactctag ggcagaaaag catggagtgc acataataag agattttgaa tacccctatt 138301 aactaaagga acaagaagag ggactgacac ctgtccttga ggagagtggg gggtgagctg 138361 aagttgaaac actggtgaaa attggatttg gacaactcaa aggtggtcat gacacttaag 138421 aaatatgctt gtctaggctg ggcgtggtgg ctcccacctg taatgccagc actttgggag 138481 gccgaggtgg gcggatcacc tgaggtcagg agttcaagac gcgcctgacc aacgtggaaa 138541 aaccccgtct ctactaaata caaaaattag atgggcgtgg tggtaggtgt ctgtaatccc 138601 aactactcag gaggctgagg caggagaatc gcttgaaccc gggaggcgga gattgcagtg 138661 aattgagatt gcgccactgc actccagcct gggtgacaga gcaagactcc atctcaaaaa 138721 aaaaaaaaaa aaaaaaaaga aatatgcttg tctaaaccct ttcccaaagc ctgtaagggc 138781 cctgtctaca ctgaccctgt ctgctcaatc acatttccta cctctttctg catgacactt 138841 cgctctaacc acacgtgcca agttttcctg tttgtttaag cattccttca ttcatccttc 138901 tcatattatt gagaacctgc tatttgccaa acactgtcat aggtgctggg agttcattgg 138961 taaagaaagc aggcaaaaac ccccacctca tgaaatgaag cttacatttt aatgggagac 139021 agaggcaata aacaagtgca tgtgtaggtc atatggtatc ttagacagtg atgagtatgt 139081 ggagaaaaat tagatggtgg ggagggcagg gagtgctgga gctagtattt taaatcggat 139141 ggtgagacag tctccctgag aaggagtact caagctatgc agaaaagact tcacatagaa 139201 agcctgagac taggccgggc tgtggtggct cacgcctgta atcctagcac tttgggaggc 139261 cgaggcaggc ggattgcctg agttcaggag ttggagacca gcctcggcaa cacagggaaa 139321 ccccatctct actaaaacaa aaaaaattag ccaggcatgg cagcatgcgc cgatagtccc 139381 agctactctg gaagctgagg caggagaatt gcttgaacct gggaagagga ggttgcagtg 139441 agccaagatc gtgccactgc actccagcct gggtgagagc taggctctgt ctccaaaaaa 139501 aaaaaaaaaa gagaaaagaa agcctgacac tacagcctta gaaagaaaga cccgatttta 139561 agattggccc tttgttggca tctaggaact tagatttttg ggaaggtttc ctccattccc 139621 tgatgtgaat ggttcatgat ccctgaactg tttgtgcaaa cagtatggtt tatggtgatt 139681 acttgctttt tcttctggga gtctgaaatc ttggtacttg ccagacagaa gctgctgaca 139741 tgaccagcca ccaataaaac ctctgggcat tgagtctcta atgagctccc ctggtagaca 139801 acatttcaca catgttgtca caacccacag acgggggcag gaagtatgtc ctgtgagact 139861 cccacaggag aggattctaa gaagcttgtg cctggtttcc tccagacttc acctcgcatg 139921 cctttctcct ttgctaattt tgcttagtac tttctcatct taatagatca taactctaat 139981 acagctacat gcagaatcct cctagtgaat cttcgaagaa atgtgtgcat ggtcttggga 140041 gcgctctgca tacaagctga gacctgaagt cagggacaag ccgtgcagta cttggctggg 140101 aggtgaaggc gtgtttgaca agtttgggta gcaggaagga ggccagtgtg gctgcagcag 140161 agggagcaag aaggaaatga gtggaagact aactagagga aagagggggt tccatagccg 140221 gggtgagagg aaagcccctg ggtttggagg gaggatctcg atcatgtttt tgaaaggacc 140281 actcccggta cagtgtccag aataagagga aatgcaggga gactcattaa ggggctgctg 140341 caggaatcca gccgagagat acggcttacg gctggtcacc aaactagctc ctgactcgac 140401 ccttctctgg attgttcttc cctgggtcat gcagagctga gttcctgggt cacctcctca 140461 cagcgcttcc ctggcgacta tatcttaagt cattctctac aacctattcg cccagtcacc 140521 taggccctct cccacacttt aattctttaa cagcattatc tctatctaaa aactattttc 140581 tttatttact ttaatgtaga attcctccct cctggctggg cgcggtggct cacaccagta 140641 atcccagcac tttgggaggc cgagacgggc agatcatgag gtcaggagat cgagaccatc 140701 ctggctaaca cggtgaaacc ccatctctac taaaaagaca aaaaattagc caggcatggt 140761 ggcacacacc tgtagtccca gctactcggg aggctgaggc aggagaatag cttgaacctg 140821 ggaggcggag gttgcagtaa gctgagattg tgccactgca ctccagcctg ggcaacagaa 140881 tgagactctg tctccaaaaa aaaaaaagaa ttcctccctc ctccactcac tcccaccaaa 140941 atgtaagttc catgagagca tttagtaggc atgcaataaa tatttgatca acaaaataaa 141001 tgaatgagcc aatgagctac atgatgcggt gacctgcttg gctggaactc tgtgaagcaa 141061 tcagcacaga tgactgccac attccaaaag gtctccagga gcagagagcc catgagcccc 141121 cttgccagcc acactaagca tccagtgcat cactgcactg ctttagctgt ttacagtaac 141181 acaaggagtc acggtaactc caggtgcgta catcttgccc ctctcttaac ttgcactggt 141241 ttatggtttc aacaatttcc aaaccccttt cctcttaccc tcccagtcat tcacgaagcc 141301 ccgcagggac cgtcgccttc ttaatgcctt tcaaattctg accccatctc tgccacctcc 141361 ttgtcactgt ctgctcaggc ctcatggctt ctcatgggga caactggtct ccctgctttt 141421 cttcaggctc tccttcaaat catgctccag actgctgaca aaggagcaat tccaacatat 141481 cttattgtgt ctacatcact cccaagcttc aagccctttc ctgggcgccc agggtcttca 141541 ggagcaagct taaactcttg agcaatgcac acggggaatt tcattatcta gctcatacct 141601 gggtgtgcct gacttaccaa gtcctcttcc acttcctcaa ctccattctc tgctgcaacc 141661 atactaatta tccacttatt gttccccaaa tattcaggca gcttcaggcc tccatccctt 141721 ctccacctgc tgtctgccta gaaagcccat ccttccctac tccccctcac ccatactcct 141781 ctggggccaa ctcgatctaa atcatatttc agacttcacc ttctccagaa aactttctct 141841 gtgcttctct tgatcagagc acctgcttca ttatgttgtg accacttatc tgctgcctgc 141901 gctatgccac agacacctat ggtccagaac cagctcttat tcaactgaaa acagtgcatg 141961 ctgcatctta ggtacttaca aataaatgct gcacaaaaca aagaggagta ctggtataga 142021 aacaagtgac acaaatcaag tagtggctag ttaaaaggac tgaggcaaaa atacccagaa 142081 cacttgttct aaacagcaaa ggcccaggtg cactggttac cacccctaca agatgactgg 142141 aatactcatt ccctgggcag tgattttttt tttttttttt tttttgagat ggaatcttgc 142201 tctgtagcta ggctggagtg cagtggcttg atctaggctc actgaaaatt ctgcctccgg 142261 ggttcaagcg attctcctcc ctcagcctcc cgagtagctg ggactatagg cgcccaccac 142321 tatgcctggc taattttttt tttttttaat ttttagcaga gatggggttt caccatgttg 142381 cccaggctgg tctcgaactc ctgagctcag gcaattcacc cgcctcggcc tcccaaagtg 142441 ctaggattac aggcgtgagc caccgcgcct ggtctgggca gtgatttttg actctttggt 142501 tcactgctgt gtcctcagaa cttaaacagt acctggcatg gtaggagatg aataaatatt 142561 tgcagaatag atgtcgaatg aatgacagac ctttcctagc actgctcagc taccgcagac 142621 tgaacctagc acatgttcca tagcccagaa tttatccctc tacctctgta atgttaataa 142681 caacagctaa catctactga gagcttaaca gatgccaggc actgttctga atgctttaca 142741 tttgcttatt tccttaactc ttcacaacaa accctttgat acaggtactg ttttaccaaa 142801 gggaagtgga gcacaatggc ccaaggtgac ccaggtagga aagagaagag ccaggcttca 142861 aacccaggca gtcttgctcc aggatctggc tcagaccaac atacacagag aaaaatggct 142921 gcacttgttc tgcctaaact tagagacctc atttggttgc tcgaacttgt ctgactttag 142981 tcaaatatag aaacatgaga cttggcatga aagtctctgt tgacactttt ttttttttta 143041 agtccagctg cctaatttat taagaacagg gcagaatttt ctgggtccag ggaaattcac 143101 cacaggatac ttccaaatca ccctgtggtg atcagagcct gcccttccct cagcatacag 143161 tatttcaccc ttcagaattt tccagaagac atttgagatg ttagagggaa gatgtgtgta 143221 acaggtccaa atgggtctag ggagaaacgc ccataaaggc aactgaggag gccgggcgca 143281 gtgggtcacg cctgtaatcc cagcactttg ggaagccaag gccggcggat cacaaggtca 143341 ggagttagag accagcctga ccaacatggt gaaaccccat ctctactaaa aatacaaaaa 143401 ttagccgggt gtggtggagg gtacctgtag tcccagctac ttgggaggct gagacagaag 143461 aatcgcttga acccaggagg cggaggttgc agtgagctga gattgtgcca ctgcactcca 143521 gcctgggcgg caagagtgaa actccgtctc aaaaaaaagg gcaactaagg agcaacccgg 143581 attcagccac cacatggctg gaaggtcact attacagaat tgaaaggaca acaatagttt 143641 ctgaccactg actactgacc accaaccata ttgcaagctg accacatgaa gtccctacca 143701 cacatctaac tcatttaatc ttcacaaggt ctctgtgagg caggaattat tacctctgct 143761 ttacagatga gggggaccat attcagaaag gctacgaaac gtgactaggg tcatacagct 143821 ggtacttgtt agagccaaga tttgaatcca tccctatccg acttccaaag cctgtgttct 143881 tgtcaccttc cagcccacct ggcctctaca catgtgctat gaaatcctcc tggtgcctgc 143941 aaagaaattt atggtgaaac ctccaaattg aagcttattt ttttagttta tatacctccc 144001 actcaggata aatagtcatc aaaatctcca aagtaaaaaa gaaagagtga gggaaaaaat 144061 ggctaaaatc ttctgatcac aagcccatct gggatctttc agatgctgaa aattccagaa 144121 ggctggatcg tcaagtttca gcttagaagt tgcaatcaga aatctttatt gaaataagtt 144181 tatttccccc cgaggactct gctggtataa cagatgagag atgagagtgc ctactaggga 144241 agaaagtggc gagaaagcga gtgatgctac tctatccggg aatctagaac aaacccacag 144301 aggatccaca gggatggagt gtgtgtatgg ggaggtgggc cagatgaaac aagaaaggca 144361 gaaagctaat ggttgttgaa attgaatgat ggacacttgg agttcattgt gccagtctat 144421 ctgcttctct gtgtgtttga aaatttcaca catgtacaca aagccatgac cctccaacaa 144481 gatgacatcc ttagatgtta atggcctggg ttccatgcct tcttagacac agcaccctta 144541 tggggagagg tcaaggctga aatcaggtgg ttcctccacc ctgtcctggt ctgttccctg 144601 ctggcccctc agcaagggtc tcagtacggc ccacagtcaa aaccctgcag caagttctgc 144661 ttttatagaa gaccttctgt aagtggttga aactactgtt tttcagaatc aggaaggagc 144721 attcaaaaga ctgctaaacc ctcaaacgag aacaagataa agaacaggct gaatcagcat 144781 gcgacaggag cctggttagt gagttgactg aggttacttg tgagaatgat gtcactgtgt 144841 ggaataagga agctggcaac accccagagc cagcgtgtgg ctgtgctggg agggagctgg 144901 ccccttagtg ccgagtaaag gacaaactcc aggcagatat ttgtcagatt gtggccagac 144961 acagtcttgt tcctttaaca ctttaaagga caaatggaaa gattagccaa gccctaatca 145021 tctgaagaag ccatgtgtct gagaagtctg agggaagata tattgaaaac gatctcagtt 145081 acgatgagac agctttgttt gatttacgca aaggcagaga cacaggtgta ctacatcctt 145141 caggaagcaa gtttagaaat aagatctgca agtcatagga tgtgacaggt ctgcaaatcc 145201 tgcaagtcac aggtcatcca cagtgggcta gtctagctgc ttcaagaatg ttgcccacga 145261 aaggtgggtc atgggttcac agagcattcc tgtgttccat aaccacaggt ttcatcccta 145321 actctggtca gcaacttttg aagtatatac caaggaccac aagttcaagg aagtgggcag 145381 aagaaagaat gtgatgcagt catgcagatc catcgccact attcaagaca cagcccagag 145441 cgccttccaa gggctttctg ttccactgag aataaaattc aaatcctcac aaaagatctc 145501 agggtgcaac aggactacct ggcctctgct catctctgtt cactcatttc ctcctggccc 145561 acctgctcta gcaatgttcc ttgaactcac caagctcatt tccactttag gacctttgca 145621 cctgcagctc gctcttgctt tgatactatt ccctaagtct ttgcatgatt acacagatca 145681 aacagatctc agaccaaagg tcgtcttctc aggcctttcc agaccattct agctaaagta 145741 gcatccccag tcactttcta ttatatcttg ttttcttttt tatatcactt gttactatct 145801 gatgttatta tttatttgat tgctttttag tgaactgtct ctctccagta gaatgtaagc 145861 tccatgaggg cagattctgg actgtctggc tcaccgctgc acccacggaa cctaggataa 145921 ggaatacctc ttgatgaata aataaccctc tggataggtc agtagctact ttcatatata 145981 tatgaaggca ttttttttga tgaggcactt tttcttaata tatgaataag caaaaaaata 146041 tagtatctat attcagtaaa aaattatttt gaaaaaacta aaatgatata aggttggggc 146101 tgtcctggag aatctaaggt atctagtcat catatatatg aaattcaact tttccattgt 146161 ttgataaatg ttcctgatga ctcatgtgtc tttagcacta gtttatctgt caaatggtca 146221 ctggtaagag aaagagcgct cgctgtgaag ggatgggatt cctgaatggg gatctggata 146281 cacagctggg tttgtgtgtt catgcatgtg cacaagcacc tgcagctcag aatctacaac 146341 caacagctct tagagtcagt gtggccttgg tatactttgt tttgtatcct ttaggggtgt 146401 gtgtctccaa ctttaacgta caagtcaatc acctggggat cttcttaaaa tgcagattcc 146461 aattccaggg tggggtctga gattttatat ttctaattag cgcttactca ggtgacatct 146521 gtgctgctgg ccagaggctc acactttggc aaatctctag tactgcaaaa ttgccgtctg 146581 agaaacatgc ctccagatta gctaagaaag gaagcgctaa ccacagggga cttggtcccc 146641 aagacccaaa aatatagaga acctaatttc aaacaagcag aaaggaactt ctctggaaag 146701 agacgactca gctttcttca tttgtaataa accaaatctg taaacactta aaatggaatc 146761 atttctaaat gctgacttgt gtgcggatct gggacactgt ttaattccag tatgggactg 146821 taaaatacat attcttttac aagtttcaca tttacaactc ttgggacatg agaaaggaga 146881 aagctttgcc aattctgtgt agcaatggaa ctaacatgga ccaatttccc ccatctaatt 146941 gtcttgctcc aggtataaaa ctagttgttt ttttttttct ggtttctcct ttgaaagttt 147001 cccatcctta ctaccaaccc cacaaccccc agccccctag tccaggctcc agggaggagc 147061 tcaagctgat gggtgagcct ctgatataat tgttctggtg agtgatgagc ctgttgtgac 147121 cttctcccac ttggctgctg accacgtgag agggcagggc tggtccagag tcctcacccg 147181 acctgagcgt ccaactgcaa ctgtatgttc cggcagcttt aataggactc tgtgctgccc 147241 gtattattca tagcagttgt taatgtgcct ctctccttgg gatcctgggg tgactgaagg 147301 gcaggactgg gcattcctgt gtcctttagc aaccttccaa tagcaatgac tggacatgat 147361 ccatcattgt ccacagcaac ctccctccca gtacacaaaa atgtcagaaa gcaaacctgg 147421 cttttttcct ccagtttttt gtgttcctta agaaKctgct ccacgtgcat cacgctgtct 147481 ccaatgcctg taaactctgc ttgctcttcc agcaaattat ccagggcaag cttagcctac 147541 aagaaacatt agaacagtcc agaagtaaac caacactcac tcacctgaag gtataggttt 147601 gttatgcttc aagaaaactc aaaattccaa aaaggattca agacttgtat atatctatat 147661 atgttaaaaa tttttttttt gcagctcact aactttcaaa

Following is a KIAA0861 genomic nucleotide sequence that includes the KIAA0861 SNP “KIAA0861-AA” (SEQ ID NO: 276).

GTGCCTTTCTGAACCCTGTGACCCAGCAGCCTCCATCAACTCGTCCTACC TGCCATGCACAGCTCCTCTGTGCCCCTGTACCTGAGCTCATGCTATTCCC TCTGCCAGGATGCCCTTCTCCTTCTCCACCAGGAGAAGAACACTTGCCAG TAAGACCCAGTTCTAATGTCACCCCTTCCTGACGGTATCAGGAAGAGTCA [G/C]TGATGGTGTTTTATGCTCCCAGAGAATTTGCCACATTGTGTTGTG ATTATTTTTCCACATCTGTCTCCCCCACTGGAATGAGAGCCTCACTCATC TTCATACCTCCCTGGTCTCTACCTGGTGCCAGAACCATCCTCAGGGCAGG GGAATGCTCAGGAAATAGATATTGAATAAAATAAGTGTATCCATCCATCC ATCCA

Following is a first KIAA0861 cDNA sequence (SEQ ID NO: 2).

KIAA0861 Coding Sequence (cDNA) Corresponding to SEQ ID NO: 2. (cDNA from positions 94 . . . 3189) cgctgaattctaggggaggatggcgcccccatcatcacgttcccagagtt ttcggggttcaaacacatcccagatgaagacttcctgaatgtcatgacct acctgactagcatccccagtgtggaggctgccagcattggattcattgtt gttatcgacagacgaagagacaagtggagctccgtaaaggcatccttgac acgaatagctgtggcatttccaggaaacttacagctcatattcatccttc gtccatctcgctttatccagaggacattcactgacattggcattaaatac tatcgaaatgagtttaaaacgaaagtgccgatcatcatggtaaactctgt ctctgaccttcacggctacatcgacaaaagccaactgacccgggaattag gggggactttggaatatcgccacggtcagtgggtaaatcaccgcactgcc atcgaaaactttgccttgaccttgaagaccactgcccagatgctgcagac gtttgggtcctgcctggccacagcagagctgcccagaagcatgctatcca cggaagaccttctcatgtcccacacaaggcagcgggacaagctgcaggat gagctgaaattacttggaaagcaggggaccacattgctgtcatgcatcca agaaccagcaaccaaatgtcccaacagcaaactcaatctcaaccaacttg agaatgtaactaccatggaaaggttattagttcaactggatgaaacagaa aaagcctttagtcacttttggtctgagcatcatctgaagcttaaccagtg cctacaactacagcattttgagcacgatttttgtaaggctaagcttgccc tggataatttgctggaagagcaagcagagtttacaggcattggagacagc gtgatgcacgtggagcagattcttaaggaacacaaaaaactggaggaaaa aagccaggagtccctggaaaaggcccagctgctggcactggttggggacc agctcatccaaagccaccattatgcagcagatgccatcaggccccggtgt gtggagctcaggcacctctgtgacgatttcatcaatggaaacaagaaaaa atgggacattttaggaaagtccttagagtttcatagacagctggacaagg tcagccaatggtgtgaggcaggaatctacctcttggcttcccaagctgta gacaagtgccagtctcgagaaggggttgatatcgccttgaacgacattgc gacattcctgggcacagtcaaggagtacccgttgctcagccccaaggagt tttacaacgagtttgagttgctgctcaccctcgatgcaaaggccaaagcc cagaaagttttgcagaggctggatgatgtccaggaaatatttcacaagag gcaagtgagtctgatgaaactggcagccaaacagactcgtccagtgcaac ctgtggccccacatcctgagtcttcaccaaaatgggtgtcatcaaaaacc agccagccctccacctcggtccctctagctcgtcctctgagaacgtctga ggaaccttatacggagacagagttgaactcccggggaaaggaagatgatg agactaaatttgaagtcaagagtgaagaaatctttgaaagccatcatgaa agggggaaccctgagctggagcagcaggccaggctcggagacctttcccc ccgcaggcgcattatacgtgacttgcttgagactgaagagatttacataa aagagattaaaagcataattgatggatatatcactccaatggattttatt tggctaaagcatctaattccagatgttcttcagaataacaaggactttct ctttgggaatattagagaactttacgaatttcacaacaggacttttctaa aagagttggaaaagtgtgctgagaaccctgaacttctggcacattgcttt ctcaagagaaaagaagatcttcagatatattttaaataccataagaatct gccccgagctagggcaatctggcaagagtgtcaagactgcgcctactttg gggtatgccagcgccaactggatcacaatctccctctttttaagtatctc aaaggaccaagccagagacttataaaataccagatgctgttgaagggtct gctggatttcgagtctcctgaagatatggagatagacccaggtgaactag gaggctcggctaaggatgggccaaagagaaccaaagattcagcattctca actgaactacaacaagctttggcagtgatagaggatttgatcaagtcctg tgagttggctgtggacctagcagcagtgactgaatgtccggacgatattg gaaaactaggcaagctgttgctgcacggccctttcagcgtctggacaatt cacaaggatcgttataaaatgaaggatttgattcgatttaaacccagcca gaggcaaatctacctatttgaaaggggaatagtgttctgtaagatacgaa tggagcctggggaccagggattatctcctcattacagcttcaagaaggcc atgaagctgatgacactttcaattcgccagcttggaagggggagccatag aaagtttgagattgccagtcgaaatggacttgagaaatacatcctgcagg cagcttcaaaagaaatcagagactgttggttttcagaaataagtaaatta ttgatggaacaacaaaataatatcaaagaccaaggaaatccacagtttga aatgagcacgagcaaaggcagtggagcaggatccggaccatggattaaaa atatggaaagagctaccactagcaaggaagacccggcctccagcacagga gggattaaaggctgctccagcagggagtttagctccatggacacctttga agactgtgaaggcgcagaagacatggaaaaggagagcagtgctctgagtc tcgcgggccttttccagtcggacgacagtcacgaaacctgttcctccaaa tctgctttcctggagaggggagaaagcagccagggagaaaaagaagaacg cgatgaggaggaaacggcgacccgcagcaccgaggaggagcgcgctgggg cgtccacgggccggctggctcctgcgggggcgacggctggtttccaggcg agggcgctgcgcccgaggacctccgcccaggagagctga

Following is a second KIAA0861 cDNA sequence (SEQ ID NO: 3).

ttgggcggagatgcctttaaaaaatcatccaccgcagcggtagaaacagt tttgtttggctttatttatacggaatggtttttcagtgaaatgctgtctt gcttaaaagaagagatgcctccccaggagctcacccggcgactggccaca gtgatcactcatgtcgatgaaattatgcagcaggaagtcagacccctgat ggcggtggagataatagaacaacttcacagacaatttgccattctttcag gaggccgaggggaggatggcgcccccatcatcacgttcccagagttttcg gggttcaaacacatcccagatgaagacttcctgaatgtcatgacctacct gactagcatccccagtgtggaggctgccagcattggattcattgttgtta tcgacagacgaagagacaagtggagctccgtaaaggcatccttgacacga atagctgtggcatttccaggaaacttacagctcatattcatccttcgtcc atctcgctttatccagaggacattcactgacattggcattaaatactatc gaaatgagtttaaaacgaaagtgccgatcatcatggtaaactctgtctct gaccttcacggctacatcgacaaaagccaactgacccgggaattaggggg gactttggaatatcgccacggtcagtgggtaaatcaccgcactgccatcg aaaactttgccttgaccttgaagaccactgcccagatgctgcagacgttt gggtcctgcctggccacagcagagctgcccagaagcatgctatccacgga agaccttctcatgtcccacacaaggcagcgggacaagctgcaggatgagc tgaaattacttggaaagcaggggaccacattgctgtcatgcatccaagaa ccagcaaccaaatgtcccaacagcaaactcaatctcaaccaacttgagaa tgtaactaccatggaaaggttattagttcaactggatgaaacagaaaaag cctttagtcacttttggtctgagcatcatctgaagcttaaccagtgccta caactacagcattttgagcacgatttttgtaaggctaagcttgccctgga taatttgctggaagagcaagcagagtttacaggcattggagacagcgtga tgcacgtggagcagattcttaaggaacacaaaaaactggaggaaaaaagc caggagcccctggaaaaggcccagctgctggcactggttggggaccagct catccaaagccaccattatgcagcagatgccatcaggccccggtgtgtgg agctcaggcacctctgtgacgatttcatcaatggaaacaagaaaaaatgg gacattttaggaaagtccttagagtttcatagacagctggacaaggtcag ccaatggtgtgaggcaggaatctacctcttggcttcccaagctgtagaca agtgccagtctcgagaaggggttgatatcgccttgaacgacattgcgaca ttcctgggcacagtcaaggagtacccgttgctcagccccaaggagtttta caacgagtttgagttgctgctcaccctcgatgcaaaggccaaagcccaga aagttttgcagaggctggatgatgtccaggaaatatttcacaagaggcaa gtgagtctgatgaaactggcagccaaacagactcgtccagtgcaacctgt ggccccacatcctgagtcttcaccaaaatgggtgtcatcaaaaaccagcc agccctccacctcggtccctctagctcgtcctctgagaacgtctgaggaa ccttatacggagacagagttgaactcccggggaaaggaagatgatgagac taaatttgaagtcaagagtgaagaaatctttgaaagccatcatgaaaggg ggaaccctgagctggagcagcaggccaggctcggagacctttccccccgc aggcgcattatacgtgacttgcttgagactgaagagatttacataaaaga gattaaaagcataattgatggatatatcactccaatggattttatttggc taaagcatctaattccagatgttcttcagaataacaaggactttctcttt gggaatattagagaactttacgaatttcacaacaggacttttctaaaaga gttggaaaagtgtgctgagaaccctgaacttctggcacattgctttctca agagaaaagaagatcttcagatatattttaaataccataagaatctgccc cgagctagggcaatctggcaagagtgtcaagactgcgcctactttggggt atgccagcgccaactggatcacaatctccctctttttaagtatctcaaag gaccaagccagagacttataaaataccagatgctgttgaagggtctgctg gatttcgagtctcctgaagatatggagatagacccaggtgaactaggagg ctcggctaaggatgggccaaagagaaccaaagattcagcattctcaactg aactacaacaagctttggcagtgatagaggatttgatcaagtcctgtgag ttggctgtggacctagcagcagtgactgaatgtccggacgatattggaaa actaggcaagctgttgctgcacggccctttcagcgtctggacaattcaca aggatcgttataaaatgaaggatttgattcgatttaaacccagccagagg caaatctacctatttgaaaggggaatagtgttctgtaagatacgaatgga gcctggggaccagggattatctcctcattacagcttcaagaagaccatga agctgatgacactttcaattcgccagcttggaagggggagccatagaaag tttgagattgccagtcgaaatggacttgagaaatacatcctgcaggcagc ttcaaaagaaatcagagactgttggttttcagaaataagtaaattattga tggaacaacaaaataatatcaaagaccaaggaaatccacagtttgaaatg agcacgagcaaaggcagtggagcaggatccggaccatggattaaaaatat ggaaagagctaccactagcaaggaagacccggcctccagcacaggaggga ttaaaggctgctccagcagggagtttagctccatggacacctttgaagac tgtgaaggcgcagaagacatggaaaaggagagcagtgctctgagtctcgc gggccttttccagtcggacgacagtcacgaaacctgttcctccaaatctg ctttcctggagaggggagaaagcagccagggagaaaaagaagaacgcgat gaggaggaaacggcgacccgcagcaccgaggaggagcgcgctggggcgtc cacgggccggctggctcctgcgggggcgacggctggtttccaggcgaggg cgctgcgcccgaggacctccgcccaggagagctgacctccctgcggacgc ccccgctcctcggctccagagcgcccgcattcccgggagaggcggtgtgg gggcccgggccctgcccagctacgcagaaagcagccggagcctcggcggc ggcagaaaggggacaaccagggcctcctccgaggagcccgaggggtgtcc tgggtgcgcgcctagctccgcacgggggacctcggagctgctctaaggcg cctgcagaggcgagcagagcccgcagcccacgccttctcgaccgcgcact tcgacattcggagccgggcaattctttgctgcgtgggcttctctgtgctc ccacggtaggatgtttagtagcacccctggcctctacccactaggtgcca ggaatgcgccaccccatcctccaccccgcccccaaatcgtgacaatcaaa aatgcctgcagacacgcccgcatttctccagggcggggtgggaatcggtt gagagccgcttagcccgagccttggaggagccggagccgctcaaacccgg cgggggccgcagactgggagctcccggtccgcctcccagcatccctgcga gcgttcatggggtgttcgtgttagtgccaagattgcttcgttgtagagag agttcgttccaagttactttctgaggtattttatgtatcgatttattagt ttttaaatgagttttgttagtttcagttgtattttatttttagttttatt agttgattttcatttctttgtagctttctggttatttaattttatagttt cagttactggtttatagttactttattttcaaagttatttagttgttcat tttcagttatttatatgtagttgttttgttttaggagttacagatgttca aattaatttgcttggaatttatttatttatttatttatttatttttcgag acggagtctcgctctgtcgcccaggctggagtgcagtggtgtgatctcgg ctcactgcaaaccgcctcccgggttcacgccattctcctgcctcagcctc ctgagtagctgggactacaggcgcccgccaccacgcccggctaatttttt atttggatttttagtagagacggggtttccccgtgttagccaggatggtc tcgatctcctgacctcgtgatccccctgcctcggcctcccaaagtgctgg gattacaggcgtgagccaccgcgcccagccggaatttatttttaattatc tttacaattattatctgagttatttaccttcatagttatttcactttatc ttattggagtttttgagttatttatttttacagtaacttatttgactatt aaacactcactggaagttcatg (4772 bp)

Following is a first KIAA0861 amino acid sequence (SEQ ID NO: 4).

MTYLTSIPSVEAASIGFIVVIDRRRDKWSSVKASLTRIAVAFPGNLQLIF ILRPSRFIQRTFTDIGIKYYRNEFKTKVPIIMVNSVSDLHGYIDKSQLTR ELGGTLEYRHGQWVNHRTAIENFALTLKTTAQMLQTFGSCLATAELPRSM LSTEDLLMSHTRQRDKLQDELKLLGKQGTTLLSCIQEPATKCPNSKLNLN QLENVTTMERLLVQLDETEKAFSHFWSEHHLKLNQCLQLQHFEHDFCKAK LALDNLLEEQAEFTGTGDSVMHVEQTLKEHKKLEEKSQESLEKAQLLALV GDQLTQSHHYAADATRPRCVELRHLCDDFTNGNKKKWDTLGKSLEFHRQL DKVSQWCEAGTYLLASQAVDKCQSREGVDTALNDTATFLGTVKEYPLLSP KEFYNEFELLLTLDAKAKAQKVLQRLDDVQETFHKRQVSLMKLAAKQTRP VQPVAPHPESSPKWVSSKTSQPSTSVPLARPLRTSEEPYTETELNSRGKE DDETKFEVKSEETFESHHERGNPELEQQARLGDLSPRRRTTRDLLETEET YTKETKSTTDGYTTPMDFTWLKHLTPDVLQNNKDFLFGNTRELYEFHNRT FLKELEKCAENPELLAHCFLKRKEDLQTYFKYHKNLPRARATWQECQDCA YFGVCQRQLDHNLPLFKYLKGPSQRLTKYQMLLKGLLDFESPEDMEIDPG ELGGSAKDGPKRTKDSAFSTELQQALAVIEDLIKSCELAVDLAAVTECPD DIGKLGKLLLHGPFSVWTIHKDRYKMKDLIRFKPSQRQIYLFERGIVFCK IRMEPGDQGLSPHYSFKKAMKLMTLSIRQLGRGSHRKFEIASRNGLEKYI LQAASKEIRDCWFSEISKLLMEQQNNIKDQGNPQFEMSTSKGSGAGSGPW IKNMERATTSKEDPASSTGGIKGCSSREFSSMDTFEDCEGAEDMEKESSA LSLAGLFQSDDSHETCSSKSAFLERGESSQGEKEERDEEETATRSTEEER AGASTGRLAPAGATAGFQARALRPRTSAQES (1031 aa)

Following is a second KIAA0861 amino acid sequence (SEQ ID NO: 5).

MLSCLKEEMPPQELTRRIATVITHVDEIMQQEVRPLMAVEIIEQLHRQFA ILSGGRGEDGAPIITFPEFSGFKHIPDEDFLNV MTYLTSTPSVEAASTGF TVVTDRRRDKWSSVKASLTRTAVAFPGNLQLTFTLRPSRFTQRTFTDTGT KYYRNEFKTKVPTTMVNSVSDLHGYTDKSQLTRELGGTLEYRHGQWVNHR TATENFALTLKTTAQMLQTFGSCLATAELPRSMLSTEDLLMSHTRQRDKL QDELKLLGKQGTTLLSCTQEPATKCPNSKLNLNQLENVTTMERLLVQLDE TEKAFSHFWSEHHLKLNQCLQLQHFEHDFCKAKLALDNLLEEQAEFTGTG DSVMHVEQTLKEHKKLEEKSQEPLEKAQLLALVGDQLTQSHHYAADATRP RCVELRHLCDDFTNGNKKKWDTLGKSLEFHRQLDKVSQWCEAGTYLLASQ AVDKCQSREGVDTALNDTATFLGTVKEYPLLSPKEFYNEFELLLTLDAKA KAQKVLQRLDDVQETFHKRQVSLMKLAAKQTRPVQPVAPHPESSPKWVSS KTSQPSTSVPLARPLRTSEEPYTETELNSRGKEDDETKFEVKSEETFESH HERGNPELEQQARLGDLSPRRRTTRDLLETEETYTKETKSTTDGYTTPMD FTWLKHLTPDVLQNNKDFLFGNTRELYEFHNRTFLKELEKCAENPELLAH CFLKRKEDLQTYFKYHKNLPRARATWQECQDCAYFGVCQRQLDHNLPLFK YLKGPSQRLTKYQMLLKGLLDFESPEDMEIDPGELGGSAKDGPKRTKDSA FSTELQQALAVIEDLIKSCELAVDLAAVTECPDDIGKLGKLLLHGPFSVW TIHKDRYKMKDLIRFKPSQRQIYLFERGIVFCKIRMEPGDQGLSPHYSFK KTMKLMTLSIRQLGRGSHRKFEIASRNGLEKYILQAASKEIRDCWFSEIS KLLMEQQNNIKDQGNPQFEMSTSKGSGAGSGPWIKNMERATTSKEDPASS TGGIKGCSSREFSSMDTFEDCEGAEDMEKESSALSLAGLFQSDDSHETCS SKSAFLERGESSQGEKEERDEEETATRSTEEERAGASTGRLAPAGATAGF QARALRPRTSAQES (1114 aa)

Following is an amino acid sequence alignment between KIAA0861 domain and SEC14 domain sequences (SEQ ID NOS 277-280, respectively in order of appearance).

Sequence 1: KIAA0861 1031 aa Sequences (1:2) Aligned. Score: 38 Sequence 2: Hs_DBS_GNEF 1108 aa Sequences (1:3) Aligned. Score: 37 Sequence 3: Mm_DBS_GNEF 1149 aa Sequences (1:4) Aligned. Score: 40 Sequence 4: Rn_DBS_GNEF  937 aa Sequences (2:3) Aligned. Score: 83 Sequences (2:4) Aligned. Score: 87 Sequences (3:4) Aligned. Score: 96 KIAA0861 ---------MTYLTSIPSVEAASIGFIVVIDRRRKDWSSVKASLTRIAVAFPGNLQLIPI  51 Hs_DBS_GNEF IPDKEFQNVMTYLTSIPSLQDAGIGFILVIDRRRDKWTSVKASVLRIAASFPANLQLVLV 180 Mm_DBS_GNEF IPDKEFQNVMTYLTSIPSLQDAGIGFILVIDRPQDKSTSVKASVLRIAASFPANLQLVLV 165 Rn_DBS_GNEF -------------------QDAGIGFILVIDRPQDKWTSVKASVLRIAASFPANLQLVLV  41                    : *.****:*****:***:*****: ***.:**.****::: KIAA0861 LRPSRFIQRTFTDIGIKYYRNEFKTKVPIIMVNSVSDLHGYIDKSQLTRELGGTLEYRHG 111 Hs_DBS_GNEF LRPTGFFQRTLSDIAFKFNRDDFKMKVPVIMLSSVPDLHGYIDKSQLTEDLGGTLDYCHS 240 Mm_DBS_GNEF LRPTGFFQRTLSDIAFKFNRDEFKMKVPVMMLSSVPELHGYIDKSQLTEDLGGTLDYCHS 225 Rn_DBS_GNEF LRPTGFFQRTLSDIAFKFNRDEFKMKVPVMMLSSVPELHGYIDKSQLTEDLGGYLDYCHS 101 ***: *:***::**.:*: *::** ***::*:.**.:***********.:*****:* *.

Following is an amino acid sequence alignment between KIAA0861 domain and SPEC domain sequences (SEQ ID NOS 281-284, respectively in order of appearance).

KIAA0861 KLLGKQGTTLLSCIQEPATKCPNSKLNLNQLENVTTMERLLVQLDETEKAFSHFWSEHHL 231 Hs_DBS_GNEF RLALKEGHSVLESLRELQAEGSEPSVNQDQLDNQATVQRLLAQLNETEAAFDEFWAKHQQ 360 Mm_DBS_GNEF QLALKEGNSILESLREPLAESAAHSVNQDQLDNQATVQPLLAQLNETEAAFDEFWAKHQQ 345 Rn_DBS_GNEF QLALTEGNSILESLREPLAESIVHSVNQDQLDNQATVKRLLTQLNETEAAFDEFWAKHQQ 221 :*  .:* ::*..::*  ::    .:* :**:* :*::***.**:*** **..**::*: KIAA0861 KLNQCLQLQHFEHDFCKAKLALDNLLEEQAEFTGIGDSVMHVEQILKEHKKLEEKSQESL 291 Hs_DBS_GNEF KLEQCLQLRHFEQGFREVKAILDAASQKIATFTDIGNSLAHVEHLLRDLASFEEKSGVAV 420 Mm_DBS_GNEF KLEQCLQLRHFEQGFREVKTTLDSMSQKIAAFTDVGNSLAHVQHLLKDLTAFEEKSSVAV 405 Rn_DBS_GNEF KLEQCLQLRHFEQGFREVKTALDSMSQKIAAFTDVGNSLAHVQHLLKDLTTFEEKSSVAV 281 **:*****:***:.* : *  **   :: * **.:*:*: **:::*::   :****  :: KIAA0861 EKAQLLALVGDQLIQSHHYAADAIRPRCVELRHLCDDFINGNKKKWDILGKSLEFHRQLD 351 Hs_DBS_GNEF ERARALSLDGEQLIGNKHYAVSDIRPKCQELRHLCDQFSAEIARRRGLLSKSLELSRRLE 480 Mm_DBS_GNEF DKARALSLEGQQLIENRHYAVDSIHPKCEELQHLCDHFASEVTRRRGLLSKSLELHSLLE 465 Rn_DBS_GNEF -TSQSPVLEGQQLIENRHYAVDISHPKCEELQHLCDHFASEVTRRRDLLSKSLELHSLLE 340   ::   * *:*** .:***.*:*:*:* **:****.*     :: .:*.****:*  *: KIAA0861 KVSQWCEAGIYLLASQAVDKCQSRESVDIALNDIATFLGRVKEYPLLSPKEFYNEFELLL 411 Hs_DBS_GNEF TSMKWCDEGIYLLASQPVDKCQSQDGAEAALQEIEKFLETGAENKIQELNAIYKSYESIL 540 Mm_DBS_GNEF TSMKWSDEGIFLLASQPVDKCQSQDGAEASFQEIEKFLETGAENKIQELNEIYKEYECIL 525 Rn_DBS_GNEF TSMKWSDEGIFLLASQPVDKCQSQDGAEAALQEIEKFLETGAENKIQELNKIYKEYECIL 400 .  :*.: **:*****.******::*.: ::::* .** *  *  : . : :*:*:* :* KIAA0861 TLDAKAKAQKVLQRLDDVQEIFHKRQVSLMKLAAKQTRPVQPVAPHPESSPKWVSSKTSQ 471 Hs_DBS_GNEF NQDLMEHVRKVFQKQASMEEVFHRRQASLKKLAARQTRPVQPVAPRPEAL-----AKSPC 595 Mm_DBS_GNEF NQDLLEHVQKVFQKQESTEEMFHRRQASLKKLAAKQTRPVQPVAPRPEAL-----TKSPS 580 Rn_DBS_GNEF NQDLLEHVQKVFQKQESTERMFHRRQASLKKLAAKQTRPVQPVAPRPEAL-----TKSPS 455 . *   :.:**:*:  . :*:**:**.** ****:**********:**:      :*:.

Following is an amino acid sequence alignment between KIAA0861 domain and RhoGEF domain sequences (SEQ ID NOS 285-288, respectively in order of appearance).

KIAA0861 GDLSPRRRIIRDLLETEEIYIKEIKSIIDGYITPMDFIWLKHLIPDVLQNNKDFLFGNIR 591 Hs_DBS_GNEF AIL--RRHVMSELLDTERAYVEELLCVLEGYAAEMDNPLMAHLLSTGLHNKKDVLFGNME 701 Mm_DBS_GNEF AIL--RRHVMNELLDTERAYVEELLCVLEGYAAEMDNPLMAHLISTGLQNKKNILFGNME 687 Rn_DBS_GNEF AIL--RRHVMNELLDTERAYVEELLCVLEGYAAEMDNPLMAHLISTGLQNKKNILFGNME 562 . *  **::: :**:**. *::*: .:::** : **   : **:.  *:*:*:.****:. KIAA0861 ELYEFHNRTFLKELEKCAENPELLAHCFLKRKEDLQIYFKYHKNLPRARAIWQECQDCAY 651 Hs_DBS_GNEF EIYHFHNRIFLRELENYTDCPELVGRCPLERMEDFQIYEKYCQNKPRSESLWRQCSDCPF 761 Mm_DBS_GNEF EIYHPHNRNIPAGVESCIDCPELVGRCFLERMEEFQIYEKYCQNKPRSESLWRQCSDCFF 747 Rn_DBS_GNEF EIYHFHNRIFLRELESCIDCPELVGRCFLERMEEFQIYEKYCQNKPRSESLWRQCSDCPF 622 *:*.**** :   :*.  : ***:.:***:* *::*** ** :* **:.::*::*.**.: KIAA0861 FGVCQRQLDHNLPLFKYLKGPSQRLIKYQMLLKGLLDFESPEDMEIDGGELGGSAKDGPK 711 Hs_DBS_GNEF FQECQRKLDHKLSLDSYLLKPVQRITKYQLLLKEML-----------------------K 798 Mm_DBS_GNEF FQECQKKLDHKLSLDSYLLKPVQRITKYQLLLKEML-----------------------K 784 Rn_DBS_GNEF FQECQKKLDHKLSLDSYLLKPVQRITKYQLLLKEML-----------------------K 659 *  **::***:*.* .**  * **: ***:*** :* KIAA0861 RTKDSAFSTELQQALAVIEDLIKSCELAVDLAAVTECPDDIGKLGKLLLHGPFSVWTIHK 771 Hs_DBS_GNEF YSRNCEGAEDLQEALSSILGILKAVNDSMHLIAITGYDGNLGDLGKLLMQGSFSVWTDHK 858 Mm_DBS_GNEF YSKHCEGAEDLQEALSSILGILKAVNDSMHLIAITGYDGNLGDLGKLLMQGSFSVWTDHK 844 Rn_DBS_GNEF YSKHCEGAEDLQEALSSILGILKAVNDSMHLIAITGYDGNLGDLGKLLMQGSFSVWTDHK 719  **..  : :**:**: * .::*: : ::.* *:*   .::*.*****::*.***** **

Following is an amino acid sequence alignment between KIAA0861 domain and PH domain sequences (SEQ ID NOS 289-292, respectively in order of appearance).

KIAA0861 DRY-KMDKLIRFKPSQRQIYLFERGIVFCKIRMEPGD-QGLSPHYSFKKAMKLMTLSIRQ 829 Hs_DBS_GNEF RGHTKVKELARFKPMQRHLFLHEKAVLFCKKREENGEGYEKAPSYSYKQSLNMAAVGITE 918 Mm_DBS_GNEF KGHTKVKELARFKPMQRHLFLHEKAVLFCKKREENGEGYEKAPSYSYKQSLNMTAVGITE 904 Rn_DBS_GNEF KGHTKVKELARFKPMQRHLFLHEKAVLFCKKREENGEGYEKAPSYSYKQSLNMTAVGITE 779   : *:*:* **** **:::*.*:.::*** * * *:    :* **:*::::: ::.* : KIAA0861 LGRGSHRKFEIASRNGLEKYILQAASKEIRDCWFSEISKLLMEQQNNIKDQGNPQ-FEMS 888 Hs_DBS_GNEF NVKGDAKKFEIWYNAREEVYIVQAPTPEIKAAWVNEIRKVLTSQLQACREASQHRALEQS 978 Mm_DBS_GNEF NVKGDTKKFEIWYNAREEVYIIQAPTPEIKAAWVNEIRKVLTSQLQACREASQHRALEQS 964 Rn_DBS_GNEF NVKGDTKKFEIWYNAREEVYIIQAPTPEIKAAWVNEIRKVLTSQLQACREASQHRALEQS 839   :*. :****  .   * **:**.: **: .*..** *:* .* :  :: .: : :* *

Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, yet these modifications and improvements are within the scope and spirit of the invention, as set forth in the claims which follow. All publications or patent documents cited in this specification are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference.

Citation of the above publications or documents is not intended as an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. U.S. patents, documents and other publications referenced herein are hereby incorporated by reference. 

1-85. (canceled)
 86. A method for identifying a subject at risk of breast cancer, which comprises detecting the presence or absence of one or more polymorphic variations associated with breast cancer in a nucleotide sequence set forth in SEQ ID NO: 1, a substantially identical sequence thereof or complement of the foregoing, in a sample from a subject, whereby the presence of a polymorphic variant associated with breast cancer is indicative of the subject being at risk of breast cancer.
 87. The method of claim 86, wherein the one or more polymorphic variations are selected from the group consisting of rs3811728, rs3811729, rs602646, rs488277, rs1629673, rs670232, rs575326, rs575386, rs684846, rs471365, rs496251, rs831246, rs831247, rs512071, rs1502761, rs681516, rs683302, rs619424, rs620722, rs529055, rs664010, rs678454, rs2653845, rs472795, rs507079, rs534333, rs535298, rs536213, rs831245, rs639690, rs684174, rs571761, rs1983421, rs4630966, rs2314415, rs6788196, rs2103062, rs9827084, rs9864865, rs6804951, rs6770548, rs1403452, rs7609994, rs9838250, rs9863404, rs903950, rs6787284, rs2017340, rs2001449, rs1317288, rs7635891, rs10704581, rs11371910, rs10937118, rs7642053, rs3821522, rs2029926, rs1390831, rs7643890, rs11925606, rs9826325, rs6800429, rs6803368, rs1353566, rs2272115, rs2272116, rs3732603, rs940055, rs2314730, rs2030578, rs2049280, rs3732602, rs2293203, rs7639705, and position 13507 of SEQ ID NO:
 1. 88. The method of claim 86, which further comprises obtaining the nucleic acid sample from the subject.
 89. The method of claim 86, wherein a polymorphic variation is detected at one or more positions in a region spanning positions 14647 to 48849 in SEQ ID NO:
 1. 90. The method of claim 86, wherein a polymorphic variation is rs4630966.
 91. The method of claim 86, wherein a polymorphic variation is rs9827084.
 92. The method of claim 86, wherein a polymorphic variation is rs9864865.
 93. The method of claim 86, wherein a polymorphic variation is rs6804951.
 94. The method of claim 86, wherein a polymorphic variation is rs6770548, rs1403452 and rs2001449.
 95. The method of claim 86, wherein one or more polymorphic variations are detected at one or more positions in linkage disequilibrium with a polymorphic variation at one or more positions selected from the group consisting of rs3811728, rs3811729, rs602646, rs488277, rs1629673, rs670232, rs575326, rs575386, rs684846, rs471365, rs496251, rs831246, rs831247, rs512071, rs1502761, rs681516, rs683302, rs619424, rs620722, rs529055, rs664010, rs678454, rs2653845, rs472795, rs507079, rs534333, rs535298, rs536213, rs831245, rs639690, rs684174, rs571761, rs1983421, rs4630966, rs2314415, rs6788196, rs2103062, rs9827084, rs9864865, rs6804951, rs6770548, rs1403452, rs7609994, rs9838250, rs9863404, rs903950, rs6787284, rs2017340, rs2001449, rs1317288, rs7635891, rs10704581, rs11371910, rs10937118, rs7642053, rs3821522, rs2029926, rs1390831, rs7643890, rs11925606, rs9826325, rs6800429, rs6803368, rs1353566, rs2272115, rs2272116, rs3732603, rs940055, rs2314730, rs2030578, rs2049280, rs3732602, rs2293203, rs7639705, and position 13507 of SEQ ID NO:
 1. 96. The method of claim 86, wherein detecting the presence or absence of the one or more polymorphic variations comprises: hybridizing an oligonucleotide to the nucleic acid sample, wherein the oligonucleotide is complementary to a nucleotide sequence in the nucleic acid and hybridizes to a region adjacent to the polymorphic variation; extending the oligonucleotide in the presence of one or more nucleotides, yielding extension products; and detecting the presence or absence of a polymorphic variation in the extension products.
 97. The method of claim 86, wherein the subject is a human.
 98. A method of genotyping a nucleic acid which comprises determining the nucleotide corresponding to position 13507 of SEQ ID NO: 1 in the nucleic acid.
 99. An isolated nucleic acid which comprises a cytosine at position 13507 of SEQ ID NO: 1, or a cytosine at a position corresponding to position 13507 of SEQ ID NO: 1 in a substantially identical nucleic acid.
 100. An oligonucleotide comprising a nucleotide sequence complementary to a portion of the nucleotide sequence of claim 99, wherein the 3′ end of the oligonucleotide is adjacent to a polymorphic variation.
 101. A microarray comprising an isolated nucleic acid of claim 99 linked to a solid support.
 102. An isolated polypeptide encoded by the isolated nucleic acid sequence of claim
 99. 103. A method of targeting information for preventing or treating breast cancer to a subject in need thereof, which comprises detecting the presence or absence of one or more polymorphic variations associated with breast cancer in SEQ ID NO: 1, a substantially identical nucleotide sequence thereof or complement of the foregoing in a nucleic acid sample from a subject, and directing information for preventing or treating breast cancer to a subject in need thereof based upon the presence or absence of the one or more polymorphic variations in the nucleic acid sample.
 104. The method of claim 103, wherein the polymorphic variation is detected in a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence in SEQ ID NO: 1, 2 or 3; (b) a nucleotide sequence which encodes a polypeptide encoded by a nucleotide sequence in SEQ ID NO: 1, 2 or 3; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to the amino acid sequence encoded by a nucleotide sequence in SEQ ID NO: 1, 2 or 3; (d) a fragment of a nucleotide sequence of (a), (b), or (c) comprising the polymorphic variation.
 105. The method of claim 103, wherein the one or more polymorphic variations are detected at one or more positions corresponding to a position selected from the group consisting of rs3811728, rs3811729, rs602646, rs488277, rs1629673, rs670232, rs575326, rs575386, rs684846, rs471365, rs496251, rs831246, rs831247, rs512071, rs1502761, rs681516, rs683302, rs619424, rs620722, rs529055, rs664010, rs678454, rs2653845, rs472795, rs507079, rs534333, rs535298, rs536213, rs831245, rs639690, rs684174, rs571761, rs1983421, rs4630966, rs2314415, rs6788196, rs2103062, rs9827084, rs9864865, rs6804951, rs6770548, rs1403452, rs7609994, rs9838250, rs9863404, rs903950, rs6787284, rs2017340, rs2001449, rs1317288, rs7635891, rs10704581, rs11371910, rs10937118, rs7642053, rs3821522, rs2029926, rs1390831, rs7643890, rs11925606, rs9826325, rs6800429, rs6803368, rs1353566, rs2272115, rs2272116, rs3732603, rs940055, rs2314730, rs2030578, rs2049280, rs3732602, rs2293203, rs7639705, and position 13507 of SEQ ID NO:
 1. 106. The method of claim 103, wherein the information comprises a description of a breast cancer detection procedure, a chemotherapeutic treatment, a surgical treatment, a radiation treatment, a preventative treatment of breast cancer, and combinations of the foregoing. 